@article{michael_chang_2024, title={A survey of C-C bond formation strategies and mechanism deployed by iron-containing enzymes in natural products}, volume={161}, ISSN={["1464-5416"]}, DOI={10.1016/j.tet.2024.134084}, abstractNote={Developing effective carbon-carbon bond forming strategies is one of the central topics for organic chemistry, boosting the development of synthetic methods towards complex molecule preparation. Meanwhile, enzymes, biocatalysts existing in nature, can facilitate a stunning array of cyclization reactions through C–C bond formation, in which these late stage ''decorations'' are essential for inherent bioactivity and pharmaceutical use. In this review, we discuss how iron-containing enzymes catalyze intramolecular C–C bond formations found in natural products. Several of these enzymatic processes serve as foundations in developing chemo-enzymatic strategies in complex molecule synthesis.}, journal={TETRAHEDRON}, author={Michael, Charalambos and Chang, Wei-chen}, year={2024}, month={Jul} }
 @article{paris_cheung_zhang_chang_liu_guo_2024, title={New Frontiers in Nonheme Enzymatic Oxyferryl Species}, volume={8}, ISSN={["1439-7633"]}, DOI={10.1002/cbic.202400307}, abstractNote={Abstract Non‐heme mononuclear iron dependent (NHM−Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2–4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high‐valent reactive iron intermediate, the S =2 oxyferryl (Fe(IV)‐oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM−Fe enzymes containing a 2‐His‐Fe or 2‐His‐1‐carboxylate‐Fe center. However, for 3‐His/4‐His‐Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S =1 Fe(IV) intermediate in a 3‐His‐Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon‐sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S =2 Fe(IV)‐oxo intermediates, the discovery of the first S =1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S =2 and the S =1 Fe(IV)‐oxo species. Finally, we postulate the possible reasons to utilize an S =1 Fe(IV) species in OvoA and their implications to other 3‐His/4‐His‐Fe enzymes.}, journal={CHEMBIOCHEM}, author={Paris, Jared C. and Cheung, Yuk Hei and Zhang, Tao and Chang, Wei-chen and Liu, Pinghua and Guo, Yisong}, year={2024}, month={Aug} }
 @article{wenger_martinie_ushimaru_pollock_sil_li_hoang_palowitch_graham_schaperdoth_et al._2024, title={Optimized Substrate Positioning Enables Switches in the C-H Cleavage Site and Reaction Outcome in the Hydroxylation-Epoxidation Sequence Catalyzed by Hyoscyamine 6<i>β</i>-Hydroxylase}, volume={8}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.4c04406}, abstractNote={Hyoscyamine 6β-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C–H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O–C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would─analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N-acetylnorloline synthase (LolO)─coordinate as alkoxide to the C7–H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C–H cleavage (C6 vs C7) and C–O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6β-hydroxyhyoscyamine suggest that a modest (∼10°) shift in the Fe–O–H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2H2O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O–H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C–H cleavage.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Wenger, Eliott S. and Martinie, Ryan J. and Ushimaru, Richiro and Pollock, Christopher J. and Sil, Debangsu and Li, Aaron and Hoang, Nhi and Palowitch, Gavin M. and Graham, Brandt P. and Schaperdoth, Irene and et al.}, year={2024}, month={Aug} }
 @article{bashiri_bulloch_bramley_davidson_stuteley_young_harris_naqvi_middleditch_schmitz_et al._2024, title={Poly-γ-glutamylation of biomolecules}, volume={15}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-024-45632-1}, abstractNote={AbstractPoly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Bashiri, Ghader and Bulloch, Esther M. M. and Bramley, William R. and Davidson, Madison and Stuteley, Stephanie M. and Young, Paul G. and Harris, Paul W. R. and Naqvi, Muhammad S. H. and Middleditch, Martin J. and Schmitz, Michael and et al.}, year={2024}, month={Feb} }
 @article{chen_chen_ruszczycky_hilovsky_hostetler_liu_zhou_chang_2024, title={Variation in Biosynthesis and Metal-Binding Properties of Isonitrile-Containing Peptides Produced by Mycobacteria versus Streptomyces}, volume={3}, ISSN={["2155-5435"]}, DOI={10.1021/acscatal.4c00645}, abstractNote={A number of bacteria are known to produce isonitrile-containing peptides (INPs) that facilitate metal transport and are important for cell survival; however, considerable structural variation is observed among INPs depending on the producing organism. While nonheme iron 2-oxoglutarate-dependent isonitrilases catalyze isonitrile formation, how the natural variation in INP structure is controlled and its implications for INP bioactivity remain open questions. Herein, total chemical synthesis is utilized with X-ray crystallographic analysis of mycobacterial isonitrilases to provide a structural model of substrate specificity that explains the longer alkyl chains observed in mycobacterial versus Streptomyces INPs. Moreover, proton NMR titration experiments demonstrate that INPs regardless of the alkyl chain length are specific for binding copper instead of zinc. These results suggest that isonitrilases may act as gatekeepers in modulating the observed biological distribution of INP structures, and this distribution may be primarily related to differing metal transport requirements among the producing strains.}, journal={ACS CATALYSIS}, author={Chen, Tzu-Yu and Chen, Jinfeng and Ruszczycky, Mark W. and Hilovsky, Dalton and Hostetler, Tyler and Liu, Xiaojing and Zhou, Jiahai and Chang, Wei-chen}, year={2024}, month={Mar} }
 @article{gering_li_tang_swartz_chang_makris_2023, title={A Ferric-Superoxide Intermediate Initiates P450-Catalyzed Cyclic Dipeptide Dimerization}, volume={8}, ISSN={["1520-5126"]}, url={https://doi.org/10.1021/jacs.3c04542}, DOI={10.1021/jacs.3c04542}, abstractNote={The cytochrome P450 (CYP) AspB is involved in the biosynthesis of the diketopiperazine (DKP) aspergilazine A. Tryptophan-linked dimeric DKP alkaloids are a large family of natural products that are found in numerous species and exhibit broad and often potent bioactivity. The proposed mechanisms for C-N bond formation by AspB, and similar C-C bond formations by related CYPs, have invoked the use of a ferryl-intermediate as an oxidant to promote substrate dimerization. Here, the parallel application of steady-state and transient kinetic approaches reveals a very different mechanism that involves a ferric-superoxide species as a primary oxidant to initiate DKP-assembly. Single turnover kinetic isotope effects and a substrate analog suggest the probable nature and site for abstraction. The direct observation of CYP-superoxide reactivity rationalizes the atypical outcome of AspB and reveals a new reaction manifold in heme enzymes.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Gering, Hannah E. and Li, Xiaojun and Tang, Haoyu and Swartz, Paul D. and Chang, Wei-Chen and Makris, Thomas M.}, year={2023}, month={Aug} }
 @article{paris_hu_wen_weitz_cheng_gee_tang_kim_vegas_chang_et al._2023, title={An <i>S</i>=1 Iron(IV) Intermediate Revealed in a Non-Heme Iron Enzyme-Catalyzed Oxidative C-S Bond Formation}, volume={9}, ISSN={["1521-3773"]}, DOI={10.1002/anie.202309362}, abstractNote={AbstractErgothioneine (ESH) and ovothiol A (OSHA) are two natural thiol‐histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2‐dependent C−S bond formation catalyzed by non‐heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four‐histidine ligand environment (three from the protein residues and one from the substrate) in enabling C−S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non‐heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C−S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure‐function relationship of high‐valent iron intermediates supported by a histidine rich ligand environment.}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Paris, Jared C. and Hu, Sha and Wen, Aiwen and Weitz, Andrew C. and Cheng, Ronghai and Gee, Leland B. and Tang, Yijie and Kim, Hyomin and Vegas, Arturo and Chang, Wei-chen and et al.}, year={2023}, month={Sep} }
 @misc{canty_chang_2023, title={Current understanding of lignan biosynthesis}, ISSN={["1551-7012"]}, DOI={10.24820/ark.5550190.p012.006}, abstractNote={Lignans constitute a large and diverse class of molecules widely distributed throughout the plant kingdom. They exhibit a wide range of pharmaceutically relevant biological activities and have attracted widespread research interests. This review covers the current understanding of lignan biosynthesis and aims to highlight key biosynthetic transformations responsible for their structural and biological diversity}, journal={ARKIVOC}, author={Canty, Nicholas Koenig and Chang, Wei-chen}, year={2023} }
 @article{phan_manley_skirboll_cha_hilovsky_chang_thompson_liu_makris_2023, title={Excision of a Protein-Derived Amine for <i>p</i>-Aminobenzoate Assembly by the Self-Sacrificial Heterobimetallic Protein CADD}, volume={62}, ISSN={["1520-4995"]}, url={https://doi.org/10.1021/acs.biochem.3c00406}, DOI={10.1021/acs.biochem.3c00406}, abstractNote={Chlamydia protein associating with death domains (CADD), the founding member of a recently discovered class of nonheme dimetal enzymes termed hemeoxygenase-like dimetaloxidases (HDOs), plays an indispensable role in pathogen survival. CADD orchestrates the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate via the self-sacrificial excision of a protein-derived tyrosine (Tyr27) and several additional processing steps, the nature and timing of which have yet to be fully clarified. Nuclear magnetic resonance (NMR) and proteomics approaches reveal the source and probable timing of amine installation by a neighboring lysine (Lys152). Turnover studies using limiting O2 have identified a para-aminobenzaldehyde (pABCHO) metabolic intermediate that is formed on the path to pABA formation. The use of pABCHO and other probe substrates shows that the heterobimetallic Fe/Mn form of the enzyme is capable of oxygen insertion to generate the pABA-carboxylate.}, number={22}, journal={BIOCHEMISTRY}, author={Phan, Han N. and Manley, Olivia M. and Skirboll, Sydney S. and Cha, Lide and Hilovsky, Dalton and Chang, Wei-chen and Thompson, Peter M. and Liu, Xiaojing and Makris, Thomas M.}, year={2023}, month={Nov}, pages={3276–3282} }
 @article{ushimaru_cha_shimo_li_paris_mori_miyamoto_coffer_uchiyama_guo_et al._2023, title={Mechanistic Analysis of Stereodivergent Nitroalkane Cyclopropanation Catalyzed by Nonheme Iron Enzymes}, volume={145}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.3c08413}, abstractNote={BelL and HrmJ are α-ketoglutarate-dependent nonheme iron enzymes that catalyze the oxidative cyclization of 6-nitronorleucine, resulting in the formation of two diastereomeric 3-(2-nitrocyclopropyl)alanine (Ncpa) products containing trans-cyclopropane rings with (1'R,2'R) and (1'S,2'S) configurations, respectively. Herein, we investigate the catalytic mechanism and stereodivergency of the cyclopropanases. The results suggest that the nitroalkane moiety of the substrate is first deprotonated to produce the nitronate form. Spectroscopic analyses and biochemical assays with substrates and analogues indicate that an iron(IV)-oxo species abstracts proS-H from C4 to initiate intramolecular C-C bond formation. A hydroxylation intermediate is unlikely to be involved in the cyclopropanation reaction. Additionally, a genome mining approach is employed to discover new homologues that perform the cyclopropanation of 6-nitronorleucine to generate cis-configured Ncpa products with (1'R,2'S) or (1'S,2'R) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.}, number={44}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Ushimaru, Richiro and Cha, Lide and Shimo, Shotaro and Li, Xiaojun and Paris, Jared C. and Mori, Takahiro and Miyamoto, Kazunori and Coffer, Lindsay and Uchiyama, Masanobu and Guo, Yisong and et al.}, year={2023}, month={Oct}, pages={24210–24217} }
 @article{cha_paris_zanella_spletzer_yao_guo_chang_2023, title={Mechanistic Studies of Aziridine Formation Catalyzed by Mononuclear Non-Heme Iron Enzymes}, volume={3}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.2c12664}, abstractNote={Aziridines are compounds with a nitrogen-containing three-membered ring. When it is incorporated into natural products, the reactivity of the strained ring often drives the biological activities of aziridines. Despite its importance, the enzymes and biosynthetic strategies deployed to install this reactive moiety remain understudied. Herein, we report the use of in silico methods to identify enzymes with potential aziridine-installing (aziridinase) functionality. To validate candidates, we reconstitute enzymatic activity in vitro and demonstrate that an iron(IV)-oxo species initiates aziridine ring closure by the C-H bond cleavage. Furthermore, we divert the reaction pathway from aziridination to hydroxylation using mechanistic probes. This observation, isotope tracing experiments using H218O and 18O2, and quantitative product analysis, provide evidence for the polar capture of a carbocation species by the amine in the pathway to aziridine installation.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Cha, Lide and Paris, Jared C. and Zanella, Brady and Spletzer, Martha and Yao, Angela and Guo, Yisong and Chang, Wei-chen}, year={2023}, month={Mar} }
 @article{wang_lu_cha_chen_palacios_li_guo_chang_chen_2023, title={Repurposing Iron- and 2-Oxoglutarate-Dependent Oxygenases to Catalyze Olefin Hydration}, volume={9}, ISSN={["1521-3773"]}, DOI={10.1002/anie.202311099}, abstractNote={AbstractMononuclear nonheme iron(II) and 2‐oxoglutarate (Fe/2OG)‐dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C−H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration—a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron‐oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG‐independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Wang, Bingnan and Lu, Yong and Cha, Lide and Chen, Tzu-Yu and Palacios, Philip M. and Li, Liping and Guo, Yisong and Chang, Wei-chen and Chen, Chuo}, year={2023}, month={Sep} }
 @article{chen_zheng_zhang_chen_cha_tang_guo_zhou_wang_liu_et al._2022, title={Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis}, volume={12}, ISSN={["2155-5435"]}, DOI={10.1021/acscatal.1c04869}, abstractNote={Despite the diversity of reactions catalyzed by 2-oxoglutarate-dependent nonheme iron (Fe/2OG) enzymes identified in recent years, only a limited number of these enzymes have been investigated in mechanistic detail. In particular, several Fe/2OG-dependent enzymes capable of catalyzing isocyanide formation have been reported. While the glycine moiety has been identified as a biosynthon for the isocyanide group, how the actual conversion is effected remains obscure. To elucidate the catalytic mechanism, we characterized two previously unidentified (AecA and AmcA) along with two known (ScoE and SfaA) Fe/2OG-dependent enzymes that catalyze N≡C triple bond installation using synthesized substrate analogues and potential intermediates. Our results indicate that isocyanide formation likely entails a two-step sequence involving an imine intermediate that undergoes decarboxylation-assisted desaturation to yield the isocyanide product. Results obtained from the in vitro experiments are further supported by mutagenesis, the product-bound enzyme structure, and in silico analysis.}, number={4}, journal={ACS CATALYSIS}, author={Chen, Tzu-Yu and Zheng, Ziyang and Zhang, Xuan and Chen, Jinfeng and Cha, Lide and Tang, Yijie and Guo, Yisong and Zhou, Jiahai and Wang, Binju and Liu, Hung-wen and et al.}, year={2022}, month={Feb}, pages={2270–2279} }
 @article{kim_chen_cha_zhou_xing_canty_zhang_chang_2022, title={Elucidation of divergent desaturation pathways in the formation of vinyl isonitrile and isocyanoacrylate}, volume={13}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-022-32870-4}, abstractNote={AbstractTwo different types of desaturations are employed by iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes to construct vinyl isonitrile and isocyanoacrylate moieties found in isonitrile-containing natural products. A substrate-bound protein structure reveals a plausible strategy to affect desaturation and hints at substrate promiscuity of these enzymes. Analogs are synthesized and used as mechanistic probes to validate structural observations. Instead of proceeding through hydroxylated intermediate as previously proposed, a plausible carbocation species is utilized to trigger C=C bond installation. These Fe/2OG enzymes can also accommodate analogs with opposite chirality and different functional groups including isonitrile-(D)-tyrosine,N-formyl tyrosine, and phloretic acid, while maintaining the reaction selectivity.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Kim, Wantae and Chen, Tzu-Yu and Cha, Lide and Zhou, Grace and Xing, Kristi and Canty, Nicholas Koenig and Zhang, Yan and Chang, Wei-Chen}, year={2022}, month={Sep} }
 @article{li_xue_guo_chang_2022, title={Mechanism of Methyldehydrofosmidomycin Maturation: Use Olefination to Enable Chain Elongation}, volume={144}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.2c01924}, abstractNote={Utilization of mononuclear iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes to enable C-H bond functionalization is a widely used strategy to diversify the structural complexity of natural products. Besides those well-studied reactions including hydroxylation, epoxidation, and halogenation, in the biosynthetic pathway of dehydrofosmidomycin, an Fe/2OG enzyme is reported to catalyze desaturation, alkyl chain elongation, along with demethylation in which trimethyl-2-aminoethylphosphonate is converted into methyldehydrofosmidomycin. How this transformation takes place is largely unknown. Herein, we characterized the reactive species, revealed the structure of the reaction intermediate, and used mechanistic probes to investigate the reaction pathway and mechanism. These results led to the elucidation of a two-step process in which the first reaction employs a long-lived Fe(IV)-oxo species to trigger C═C bond installation. During the second reaction, the olefin installed in situ enables C-C bond formation that is accompanied with a C-N bond cleavage and hydroxylation to furnish the alkyl chain elongation and demethylation. This work expands the reaction repertoire of Fe/2OG enzymes by introducing a new pathway to the known C-C bond formation mechanisms utilized by metalloenzymes.}, number={18}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Li, Xiaojun and Xue, Shan and Guo, Yisong and Chang, Wei-Chen}, year={2022}, month={May}, pages={8257–8266} }
 @article{manley_phan_stewart_mosley_xue_cha_bai_lightfoot_rucker_collins_et al._2022, title={Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para-aminobenzoate in Chlamydia trachomatis}, volume={119}, ISSN={["1091-6490"]}, url={http://dx.doi.org/10.1073/pnas.2210908119}, DOI={10.1073/pnas.2210908119}, abstractNote={
            Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of
            para
            -aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen
            Chlamydia trachomatis
            . The pathway used by
            Chlamydiae
            for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from
            l
            -tyrosine. As a member of the emerging superfamily of heme oxygenase–like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is ∼14 Å from the dimetal site. We propose that this self-sacrificial reaction occurs through O
            2
            activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the “substrate” Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in
            C. trachomatis
            , which will inform the design of novel therapeutics.
          }, number={39}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Manley, Olivia M. and Phan, Han N. and Stewart, Allison K. and Mosley, Dontae A. and Xue, Shan and Cha, Lide and Bai, Hongxia and Lightfoot, Veda C. and Rucker, Pierson A. and Collins, Leonard and et al.}, year={2022}, month={Sep} }
 @article{chen_chang_2021, title={A postreplicative C5-cytosine hypermodification triggered by bacteriophage methyltransferase and hydroxylase}, volume={118}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.2109992118}, abstractNote={Viruses of bacteria, also known as bacteriophages, harbor the greatest diversity of DNA modifications identified to date. To fight against restriction endo-nucleases of their hosts, bacteriophages modify their genomic DNA through introduction of various moieties including amino acids, polyamines, and sugars (1, 2). A series of transformations are involved in DNA base modification. Followed by formation of hydroxymethyl pyrimidine nucleotides, which are utilized by DNA polymerase, replication and postreplicative modifications furnish installation of these moieties onto the DNA poly-mer (3 – 7). Burke et al. (8) show that bacteriophages re-sort to C5-cytosine methyltransferase (C5-MT) and 5-methylcytosine dioxygenase ten-eleven translocation enzyme (TET) as an alternative mechanism to postrepli-catively form hydroxymethylcytosine on DNA. The bacteriophage TET enables site-specific hydroxylation of 5-methylcytosine (5mC), installed by C5-MT, to produce 5-hydroxymethylcytosine (5hmC). Through bioinfor-matic screening, the authors identify and characterize tailoring enzymes, such as glycosyltransferases, that collaborate with phage C5-MT and TET to further elaborate DNA at 5hmC site.}, number={28}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Chen, Tzu-Yu and Chang, Wei-chen}, year={2021}, month={Jul} }
 @article{manley_tang_xue_guo_chang_makris_2021, title={BesC Initiates C-C Cleavage through a Substrate-Triggered and Reactive Diferric-Peroxo Intermediate}, volume={12}, ISSN={["1520-5126"]}, url={https://doi.org/10.1021/jacs.1c11109}, DOI={10.1021/jacs.1c11109}, abstractNote={BesC catalyzes the iron- and O2-dependent cleavage of 4-chloro-l-lysine to form 4-chloro-l-allylglycine, formaldehyde, and ammonia. This process is a critical step for a biosynthetic pathway that generates a terminal alkyne amino acid which can be leveraged as a useful bio-orthogonal handle for protein labeling. As a member of an emerging family of diiron enzymes that are typified by their heme oxygenase-like fold and a very similar set of coordinating ligands, recently termed HDOs, BesC performs an unusual type of carbon-carbon cleavage reaction that is a significant departure from reactions catalyzed by canonical dinuclear-iron enzymes. Here, we show that BesC activates O2 in a substrate-gated manner to generate a diferric-peroxo intermediate. Examination of the reactivity of the peroxo intermediate with a series of lysine derivatives demonstrates that BesC initiates this unique reaction trajectory via cleavage of the C4-H bond; this process represents the rate-limiting step in a single turnover reaction. The observed reactivity of BesC represents the first example of a dinuclear-iron enzyme that utilizes a diferric-peroxo intermediate to capably cleave a C-H bond as part of its native function, thus circumventing the formation of a high-valent intermediate more commonly associated with substrate monooxygenations.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, publisher={American Chemical Society (ACS)}, author={Manley, Olivia M. and Tang, Haoyu and Xue, Shan and Guo, Yisong and Chang, Wei-chen and Makris, Thomas M.}, year={2021}, month={Dec} }
 @article{tang_tang_kurnikov_liao_chan_kurnikova_guo_chang_2021, title={Harnessing the Substrate Promiscuity of Dioxygenase AsqJ and Developing Efficient Chemoenzymatic Synthesis for Quinolones}, volume={11}, ISSN={["2155-5435"]}, DOI={10.1021/acscatal.1c01150}, abstractNote={Nature has developed complexity-generating reactions within natural product biosynthetic pathways. However, direct utilization of these pathways to prepare compound libraries remains challenging due to limited substrate scopes, involvement of multiple-step reactions, and moderate robustness of these sophisticated enzymatic transformations. Synthetic chemistry, on the other hand, offers an alternative approach to prepare natural product analogs. However, owing to complex and diverse functional groups appended on the targeted molecules, dedicated design and development of synthetic strategies are typically required. Herein, by leveraging the power of chemo-enzymatic synthesis, we report an approach to bridge the gap between biological and synthetic strategies in the preparation of quinolone alkaloid analogs. Leading by in silico analysis, the predicted substrate analogs were chemically synthesized. The AsqJ-catalyzed asymmetric epoxidation of these substrate analogues was followed by an Lewis Acid-triggered ring contraction to complete the viridicatin formation. We evaluated the robustness of this method in gram-scale reactions. Lastly, through chemoenzymatic cascades, a library of quinolone alkaloids is effectively prepared.}, number={12}, journal={ACS CATALYSIS}, author={Tang, Haoyu and Tang, Yijie and Kurnikov, Igor V and Liao, Hsuan-Jen and Chan, Nei-Li and Kurnikova, Maria G. and Guo, Yisong and Chang, Wei-chen}, year={2021}, month={Jun}, pages={7186–7192} }
 @article{li_shimaya_dairi_chang_ogasawara_2021, title={Identification of Cyclopropane Formation in the Biosyntheses of Hormaomycins and Belactosins: Sequential Nitration and Cyclopropanation by Metalloenzymes}, volume={12}, ISSN={["1521-3773"]}, DOI={10.1002/anie.202113189}, abstractNote={AbstractHormaomycins and belactosins are peptide natural products that contain unusual cyclopropane moieties. Bioinformatics analysis of the corresponding biosynthetic gene clusters showed that two conserved genes,hrmI/belKandhrmJ/belL, were potential candidates for catalyzing cyclopropanation. Using in vivo and in vitro assays, the functions of HrmI/BelK and HrmJ/BelL were established. HrmI and BelK, which are heme oxygenase‐like dinuclear iron enzymes, catalyze oxidation of the ϵ‐amino group ofl‐lysine to affordl‐6‐nitronorleucine. Subsequently, HrmJ and BelL, which are iron‐ and α‐ketoglutarate‐dependent oxygenases, effectively convertl‐6‐nitronorleucine into 3‐(trans‐2‐nitrocyclopropyl)‐alanine through C4−C6 bond installation. These observations disclose a novel pathway of cyclopropane ring construction and exemplify the new chemistry involving metalloenzymes in natural product biosynthesis.}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Li, Xiaojun and Shimaya, Ryo and Dairi, Tohru and Chang, Wei-chen and Ogasawara, Yasushi}, year={2021}, month={Dec} }
 @article{tang_wu_lin_han_tu_yang_chien_chan_chang_2022, title={Mechanistic analysis of carbon-carbon bond formation by deoxypodophyllotoxin synthase}, volume={119}, ISSN={["1091-6490"]}, DOI={10.1073/pnas.2113770119}, abstractNote={Significance
          The completion of the tetracyclic core of etoposide, classified by the World Health Organization as an essential medicine, by the Fe/2OG oxygenase deoxypodophyllotoxin synthase follows a hybrid radical-polar pathway not previously seen in other members of this enzyme class. The implication of a substrate-based benzylic carbocation in this mechanism will inform ongoing efforts to create analogs of this important drug with improved or emergent properties and represents a new route for resolution of the initial substrate radical that is common to members of the class. This study adds to our understanding on a growing number of biochemical transformations in which carbocation intermediates are likely to be crucial.}, number={1}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Tang, Haoyu and Wu, Min-Hao and Lin, Hsiao-Yu and Han, Meng-Ru and Tu, Yueh-Hua and Yang, Zhi-Jie and Chien, Tun-Cheng and Chan, Nei-Li and Chang, Wei-chen}, year={2022}, month={Jan} }
 @article{cha_milikisiyants_davidson_xue_smirnova_smirnov_guo_chang_2020, title={Alternative Reactivity of Leucine 5-Hydroxylase Using an Olefin-Containing Substrate to Construct a Substituted Piperidine Ring}, volume={59}, ISSN={["0006-2960"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85085904478&partnerID=MN8TOARS}, DOI={10.1021/acs.biochem.0c00289}, abstractNote={Applying enzymatic reactions to produce useful molecules is a central focus of chemical biology. Iron and 2-oxoglutarate (Fe/2OG) enzymes are found in all kingdoms of life and catalyze a broad array of oxidative transformations. Herein, we demonstrate that the activity of an Fe/2OG enzyme can be redirected when changing the targeted carbon hybridization from sp3 to sp2. During leucine 5-hydroxylase catalysis, installation of an olefin group onto the substrate redirects the Fe(IV)-oxo species reactivity from hydroxylation to asymmetric epoxidation. The resulting epoxide subsequently undergoes intramolecular cyclization to form the substituted piperidine, 2S,5S-hydroxypipecolic acid.}, number={21}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Cha, Lide and Milikisiyants, Sergey and Davidson, Madison and Xue, Shan and Smirnova, Tatyana I and Smirnov, Alex I and Guo, Yisong and Chang, Wei-Chen}, year={2020}, month={Jun}, pages={1961–1965} }
 @article{cha_chang_2020, title={An Effective Strategy to Introduce Carbon Isotopes by Simple Swaps of CO2}, volume={2}, ISSN={["2589-5974"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85094605905&partnerID=MN8TOARS}, DOI={10.1016/j.trechm.2020.10.004}, abstractNote={A novel strategy to introduce C-isotopes at the carboxylate group has been discovered. Through ‘simple’ CO2 exchange under mild conditions, the C-isotope can be incorporated effectively and has been demonstrated in a variety of biologically important molecules. Also, this methodology is used to install a C–C bond when alternative electrophiles are employed. A novel strategy to introduce C-isotopes at the carboxylate group has been discovered. Through ‘simple’ CO2 exchange under mild conditions, the C-isotope can be incorporated effectively and has been demonstrated in a variety of biologically important molecules. Also, this methodology is used to install a C–C bond when alternative electrophiles are employed.}, number={12}, journal={TRENDS IN CHEMISTRY}, author={Cha, Lide and Chang, Wei-chen}, year={2020}, month={Dec}, pages={1040–1042} }
 @article{chen_chen_tang_zhou_guo_chang_2021, title={Current Understanding toward Isonitrile Group Biosynthesis and Mechanism}, volume={39}, ISSN={["1614-7065"]}, DOI={10.1002/cjoc.202000448}, abstractNote={AbstractIsonitrile group has been identified in many natural products. Due to the broad reactivity of N≡C triple bond, these natural products have valuable pharmaceutical potentials. This review summarizes the current biosynthetic pathways and the corresponding enzymes that are responsible for isonitrile‐containing natural product generation. Based on the strategies utilized, two fundamentally distinctive approaches are discussed. In addition, recent progress in elucidating isonitrile group formation mechanisms is also presented.}, number={2}, journal={CHINESE JOURNAL OF CHEMISTRY}, author={Chen, Tzu-Yu and Chen, Jinfeng and Tang, Yijie and Zhou, Jiahai and Guo, Yisong and Chang, Wei-chen}, year={2021}, month={Feb}, pages={463–472} }
 @article{chen_xue_tsai_chien_guo_chang_2021, title={Deciphering Pyrrolidine and Olefin Formation Mechanism in Kainic Acid Biosynthesis}, volume={11}, ISSN={["2155-5435"]}, url={http://dx.doi.org/10.1021/acscatal.0c03879}, DOI={10.1021/acscatal.0c03879}, abstractNote={Metalloenzyme-catalyzed cyclization involving C–H bond activation is a powerful strategy to construct molecular complexity found in natural product biosynthesis. In the isodomoic acid and kainic ac...}, number={1}, journal={ACS CATALYSIS}, publisher={American Chemical Society (ACS)}, author={Chen, Tzu-Yu and Xue, Shan and Tsai, Wei-Chih and Chien, Tun-Cheng and Guo, Yisong and Chang, Wei-chen}, year={2021}, month={Jan}, pages={278–282} }
 @article{li_liao_tang_huang_cha_lin_lee_kurnikov_kurnikova_chang_et al._2020, title={Epoxidation Catalyzed by the Nonheme Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate}, volume={142}, ISSN={["1520-5126"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85083666781&partnerID=MN8TOARS}, DOI={10.1021/jacs.0c00484}, abstractNote={Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by non-heme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the non-heme iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogs with different para substituents ranging from electron-donating groups (e.g. methoxy) to electron-withdrawing groups (e.g. trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations and molecular dynamic simulations collectively revealed the following mechanistic insights: 1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); 2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from non-heme Fe(IV)-oxo synthetic model complexes; 3) The OAT step most likely proceeds through a step-wise process with the initial formation of C(benzylic)-O bond to generate an Fe(III)-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.}, number={13}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Li, Jikun and Liao, Hsuan-Jen and Tang, Yijie and Huang, Jhih-Liang and Cha, Lide and Lin, Te-Sheng and Lee, Justin L. and Kurnikov, Igor V and Kurnikova, Maria G. and Chang, Wei-Chen and et al.}, year={2020}, month={Apr}, pages={6268–6284} }
 @article{chen_chen_tang_zhou_guo_chang_2020, title={Pathway from N-Alkylglycine to Alkylisonitrile Catalyzed by Iron(II) and 2-Oxoglutarate-Dependent Oxygenases}, volume={59}, ISSN={["1521-3773"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85081226966&partnerID=MN8TOARS}, DOI={10.1002/anie.201914896}, abstractNote={AbstractN‐alkylisonitrile, a precursor to isonitrile‐containing lipopeptides, is biosynthesized by decarboxylation‐assisted ‐N≡C group (isonitrile) formation by using N‐alkylglycine as the substrate. This reaction is catalyzed by iron(II) and 2‐oxoglutarate (Fe/2OG) dependent enzymes. Distinct from typical oxygenation or halogenation reactions catalyzed by this class of enzymes, installation of the isonitrile group represents a novel reaction type for Fe/2OG enzymes that involves a four‐electron oxidative process. Reported here is a plausible mechanism of three Fe/2OG enzymes, Sav607, ScoE and SfaA, which catalyze isonitrile formation. The X‐ray structures of iron‐loaded ScoE in complex with its substrate and the intermediate, along with biochemical and biophysical data reveal that ‐N≡C bond formation involves two cycles of Fe/2OG enzyme catalysis. The reaction starts with an FeIV‐oxo‐catalyzed hydroxylation. It is likely followed by decarboxylation‐assisted desaturation to complete isonitrile installation.}, number={19}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Chen, Tzu-Yu and Chen, Jinfeng and Tang, Yijie and Zhou, Jiahai and Guo, Yisong and Chang, Wei-chen}, year={2020}, month={May}, pages={7367–7371} }
 @article{rajkovich_pandelia_mitchell_chang_zhang_boal_krebs_bollinger_2019, title={A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases}, volume={58}, ISSN={["0006-2960"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85062878672&partnerID=MN8TOARS}, DOI={10.1021/acs.biochem.9b00044}, abstractNote={The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, ( R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [( R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.}, number={12}, journal={BIOCHEMISTRY}, author={Rajkovich, Lauren J. and Pandelia, Maria-Eirini and Mitchell, Andrew J. and Chang, Wei-chen and Zhang, Bo and Boal, Amie K. and Krebs, Carsten and Bollinger, J. Martin, Jr.}, year={2019}, month={Mar}, pages={1627–1647} }
 @article{davidson_mcnamee_fan_guo_chang_2019, title={Repurposing Nonheme Iron Hydroxylases To Enable Catalytic Nitrile Installation through an Azido Group Assistance}, volume={141}, ISSN={["0002-7863"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85062294563&partnerID=MN8TOARS}, DOI={10.1021/jacs.8b13906}, abstractNote={Three mononuclear nonheme iron and 2-oxoglutarate dependent enzymes, l-Ile 4-hydroxylase, l-Leu 5-hydroxylase and polyoxin dihydroxylase, are previously reported to catalyze the hydroxylation of l-isoleucine, l-leucine, and l-α-amino-δ-carbamoylhydroxyvaleric acid (ACV). In this study, we showed that these enzymes can accommodate leucine isomers and catalyze regiospecific hydroxylation. On the basis of these results, as a proof-of-concept, we demonstrated that the outcome of the reaction can be redirected by installation of an assisting group within the substrate. Specifically, instead of canonical hydroxylation, these enzymes can catalyze non-native nitrile group installation when an azido group is introduced. The reaction is likely to proceed through C-H bond activation by an Fe(IV)-oxo species, followed by azido-directed C≡N bond formation. These results offer a unique opportunity to investigate and expand the reaction repertoire of Fe/2OG enzymes.}, number={8}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Davidson, Madison and McNamee, Meredith and Fan, Ruixi and Guo, Yisong and Chang, Wei-chen}, year={2019}, month={Feb}, pages={3419–3423} }
 @article{thibodeaux_chang_liu_2019, title={Unraveling flavoenzyme reaction mechanisms using flavin analogues and linear free energy relationships}, volume={620}, ISSN={["0076-6879"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85064058620&partnerID=MN8TOARS}, DOI={10.1016/bs.mie.2019.03.010}, abstractNote={Flavoenzymes mediate a large number of different chemical transformations that employ the flavin coenzyme for many different purposes. Flavins are commonly utilized in a variety of both 1- and 2-electron transfers and reactions involving oxygen activation. In addition, flavins have also been shown to function as acid/base catalysts or nucleophilic catalysts, to electrostatically stabilize transition states, and to serve simply as structural components in some enzymes. In all of these functions, the electronic properties of the flavin play a critical role. Studies carried out over a number of years have shown that these electronic properties (and subsequently, the catalytic properties of the flavoenzyme) can be manipulated by altering the substituents on the isoalloxazine ring system of the flavin. Here, we detail methods for the chemoenzymatic preparation and purification of flavin analogues, the reconstitution of apo-flavoenzymes with these analogues, and the use of linear free energy relationships (LFERs) to correlate the perturbations induced by the altered substituents. Using examples from the literature, we highlight how the use of flavin analogues and LFERs can provide mechanistic insight into the transition state structures that define flavoenyzme chemical mechanisms.}, journal={NEW APPROACHES FOR FLAVIN CATALYSIS}, author={Thibodeaux, Christopher J. and Chang, Wei-chen and Liu, Hung-wen}, year={2019}, pages={167–188} }
 @article{yu_tang_cha_milikisiyants_smirnova_smirnov_guo_chang_2018, title={Elucidating the Reaction Pathway of Decarboxylation-Assisted Olefination Catalyzed by a Mononuclear Non-Heme Iron Enzyme}, volume={140}, ISSN={["1520-5126"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85056414780&partnerID=MN8TOARS}, DOI={10.1021/jacs.8b10077}, abstractNote={Installation of olefins into molecules is a key transformation in organic synthesis. The recently discovered decarboxylation-assisted olefination in the biosynthesis of rhabduscin by a mononuclear non-heme iron enzyme ( P.IsnB) represents a novel approach in olefin construction. This method is commonly employed in natural product biosynthesis. Herein, we demonstrate that a ferryl intermediate is used for C-H activation at the benzylic position of the substrate. We further establish that P.IsnB reactivity can be switched from olefination to hydroxylation using electron-withdrawing groups appended on the phenyl moiety of the analogues. These experimental observations imply that a pathway involving an initial C-H activation followed by a benzylic carbocation species or by electron transfer coupled β-scission is likely utilized to complete C═C bond formation.}, number={45}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Yu, Cheng-Ping and Tang, Yijie and Cha, Lide and Milikisiyants, Sergey and Smirnova, Tatyana I. and Smirnov, Alex I. and Guo, Yisong and Chang, Wei-chen}, year={2018}, month={Nov}, pages={15190–15193} }
 @article{liao_li_huang_davidson_kurnikov_lin_lee_kurnikova_guo_chan_et al._2018, title={Insights into the Desaturation of Cyclopeptin and its C3 Epimer Catalyzed by a non-Heme Iron Enzyme: Structural Characterization and Mechanism Elucidation}, volume={57}, ISSN={["1521-3773"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85040694556&partnerID=MN8TOARS}, DOI={10.1002/anie.201710567}, abstractNote={AbstractAsqJ, an iron(II)‐ and 2‐oxoglutarate‐dependent enzyme found in viridicatin‐type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short‐lived FeIV‐oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ‐catalyzed desaturation.}, number={7}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Liao, Hsuan-Jen and Li, Jikun and Huang, Jhih-Liang and Davidson, Madison and Kurnikov, Igor and Lin, Te-Sheng and Lee, Justin L. and Kurnikova, Maria and Guo, Yisong and Chan, Nei-Li and et al.}, year={2018}, month={Feb}, pages={1831–1835} }
 @article{pan_bhardwaj_zhang_chang_schardl_krebs_grossman_bollinger_2018, title={Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions}, volume={57}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/ACS.BIOCHEM.8B00157}, DOI={10.1021/ACS.BIOCHEM.8B00157}, abstractNote={The core of the loline family of insecticidal alkaloids is the bicyclic pyrrolizidine unit with an additional strained ether bridge between carbons 2 and 7. Previously reported genetic and in vivo biochemical analyses showed that the presumptive iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, LolO, is required for installation of the ether bridge upon the pathway intermediate, 1-exo-acetamidopyrrolizidine (AcAP). Here we show that LolO is, in fact, solely responsible for this biosynthetic four-electron oxidation. In sequential 2OG- and O2-consuming steps, LolO removes hydrogens from C2 and C7 of AcAP to form both carbon–oxygen bonds in N-acetylnorloline (NANL), the precursor to all other lolines. When supplied with substoichiometric 2OG, LolO only hydroxylates AcAP. At higher 2OG:AcAP ratios, the enzyme further processes the alcohol to the tricyclic NANL. Characterization of the alcohol intermediate by mass spectrometry and nuclear magnetic resonance spectroscopy shows that it is 2-endo-hydroxy-1-exo-acetamidopyrrolizidine (2-endo-OH-AcAP). Kinetic and spectroscopic analyses of reactions with site-specifically deuteriated AcAP substrates confirm that the C2–H bond is cleaved first and that the responsible intermediate is, as expected, an FeIV–oxo (ferryl) complex. Analyses of the loline products from cultures fed with stereospecifically deuteriated AcAP precursors, proline and aspartic acid, establish that LolO removes the endo hydrogens from C2 and C7 and forms both new C–O bonds with retention of configuration. These findings delineate the pathway to an important class of natural insecticides and lay the foundation for mechanistic dissection of the chemically challenging oxacyclization reaction.}, number={14}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Pan, Juan and Bhardwaj, Minakshi and Zhang, Bo and Chang, Wei-chen and Schardl, Christopher L. and Krebs, Carsten and Grossman, Robert B. and Bollinger, J. Martin, Jr.}, year={2018}, month={Mar}, pages={2074–2083} }
 @article{chang_liu_guo_2018, title={Mechanistic Elucidation of Two Catalytically Versatile Iron(II)- and -Ketoglutarate-Dependent Enzymes: Cases Beyond Hydroxylation}, volume={38}, ISSN={["1548-9574"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85053453956&partnerID=MN8TOARS}, DOI={10.1080/02603594.2018.1509856}, abstractNote={Iron(II)- and α-ketoglutarate-dependent (Fe/αKG) enzymes catalyze a large array of reactions. Although hydroxylation reaction catalyzed by these enzymes has been investigated in great details, involving the ferryl (FeIV=O) as a key reactive intermediate. The mechanisms of reactions other than hydroxylation are still largely unknown. By using a combined biochemical, bio-organic, and spectroscopic approach, we have studied the mechanisms of two newly discovered Fe/αKG enzymes, FtmOx1 (endoperoxidase) and AsqJ (desaturase/epoxidase), revealing their strategies in controlling reactivity, namely the effect of redox/polar residues near the iron center, the electronic properties of the substrate, and the intrinsic reactivity of the ferryl intermediate.}, number={4}, journal={COMMENTS ON INORGANIC CHEMISTRY}, author={Chang, Wei-chen and Liu, Pinghua and Guo, Yisong}, year={2018}, pages={127–165} }
 @article{huang_tang_yu_sanyal_jia_liu_guo_chang_2018, title={Mechanistic Investigation of Oxidative Decarboxylation Catalyzed by Two Iron(II)- and 2-Oxoglutarate-Dependent Enzymes}, volume={57}, ISSN={["0006-2960"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85044655678&partnerID=MN8TOARS}, DOI={10.1021/acs.biochem.8b00115}, abstractNote={Two non-heme iron enzymes, IsnB and AmbI3, catalyze a novel decarboxylation-assisted olefination to produce indole vinyl isonitrile, an important building block for many natural products. Compared to other reactions catalyzed by this enzyme family, decarboxylation-assisted olefination represents an attractive biosynthetic route and a mechanistically unexplored pathway in constructing a C═C bond. Using mechanistic probes, transient state kinetics, reactive intermediate trapping, spectroscopic characterizations, and product analysis, we propose that both IsnB and AmbI3 initiate stereoselective olefination via a benzylic C-H bond activation by an Fe(IV)-oxo intermediate, and the reaction likely proceeds through a radical- or carbocation-induced decarboxylation to complete C═C bond installation.}, number={12}, journal={BIOCHEMISTRY}, author={Huang, Jhih-Liang and Tang, Yijie and Yu, Cheng-Ping and Sanyal, Dev and Jia, Xinglin and Liu, Xinyu and Guo, Yisong and Chang, Wei-chen}, year={2018}, month={Mar}, pages={1838–1841} }
 @article{chang_yang_tu_chien_2019, title={Reaction Mechanism of a Nonheme Iron Enzyme Catalyzed Oxidative Cyclization via C-C Bond Formation}, volume={21}, ISSN={["1523-7052"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85058840946&partnerID=MN8TOARS}, DOI={10.1021/acs.orglett.8b03670}, abstractNote={A complementary study including design of mechanistic probes, biochemical assays, model analysis, and liquid chromatography coupled mass spectrometry was conducted to establish the reaction mechanism for a nonheme iron enzyme catalyzed (-)-podophyllotoxin formation. Our results indicate that the originally proposed hydroxylated intermediate is unlikely to be involved in this reaction. Instead, the formation of benzylic radical/carbocation intermediate can be utilized to trigger the C-C bond formation to construct the C-ring of (-)-podophyllotoxin.}, number={1}, journal={ORGANIC LETTERS}, author={Chang, Wei-chen and Yang, Zhi-Jie and Tu, Yueh-Hua and Chien, Tun-Cheng}, year={2019}, month={Jan}, pages={228–232} }
 @article{rose_ghosh_maggiolo_pollock_blaesi_hajj_wei_rajakovich_chang_han_et al._2018, title={Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class i Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor}, volume={57}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85046676231&partnerID=MN8TOARS}, DOI={10.1021/acs.biochem.8b00247}, abstractNote={A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its β subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj β has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj β directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) βs. This tendency makes Fj β unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj β is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-β.}, number={18}, journal={Biochemistry}, author={Rose, H.R. and Ghosh, M.K. and Maggiolo, A.O. and Pollock, C.J. and Blaesi, E.J. and Hajj, V. and Wei, Y. and Rajakovich, L.J. and CHANG, WEI-CHEN and Han, Y. and et al.}, year={2018}, pages={2679–2693} }
 @article{ushimaru_ruszczycky_chang_yan_liu_liu_2018, title={Substrate Conformation Correlates with the Outcome of Hyoscyamine 6β-Hydroxylase Catalyzed Oxidation Reactions}, volume={140}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JACS.8B03729}, DOI={10.1021/JACS.8B03729}, abstractNote={Hyoscyamine 6β-hydroxylase (H6H) is an α-ketoglutarate dependent mononuclear nonheme iron enzyme that catalyzes C6-hydroxylation of hyoscyamine and oxidative cyclization of the resulting product to give the oxirane natural product scopolamine. Herein, the chemistry of H6H is investigated using hyoscyamine derivatives with modifications at the C6 or C7 position as well as substrate analogues possessing a 9-azabicyclo[3.3.1]nonane core. Results indicate that hydroxyl rebound is unlikely to take place during the cyclization reaction and that the hydroxylase versus oxidative cyclase activity of H6H is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted.}, number={24}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Ushimaru, Richiro and Ruszczycky, Mark W. and Chang, Wei-chen and Yan, Feng and Liu, Yung-nan and Liu, Hung-wen}, year={2018}, month={Jun}, pages={7433–7436} }
 @article{dunham_chang_mitchell_martinie_zhang_bergman_rajakovich_wang_silakov_krebs_et al._2018, title={Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of alpha-Heteroatom Assistance}, volume={140}, ISSN={["0002-7863"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85046656717&partnerID=MN8TOARS}, DOI={10.1021/jacs.8b01933}, abstractNote={Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (2H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors 1H or 2H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors 2H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.}, number={23}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Dunham, Noah P. and Chang, Wei-chen and Mitchell, Andrew J. and Martinie, Ryan J. and Zhang, Bo and Bergman, Jonathan A. and Rajakovich, Lauren J. and Wang, Bo and Silakov, Alexey and Krebs, Carsten and et al.}, year={2018}, month={Jun}, pages={7116–7126} }
 @article{chang_sanyal_huang_ittiamornkui_zhu_liu_2017, title={In Vitro Stepwise Reconstitution of Amino Acid Derived Vinyl Isocyanide Biosynthesis: Detection of an Elusive Intermediate}, volume={19}, ISSN={["1523-7052"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85014520010&partnerID=MN8TOARS}, DOI={10.1021/acs.orglett.7b00258}, abstractNote={In vitro reconstitution of a newly discovered isonitrile synthase (AmbI1 and AmbI2) and the detection of an elusive intermediate (S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1 in indolyl vinyl isocyanide biogenesis are reported. The characterization of iron/2-oxoglutarate (Fe/2OG) dependent desaturases IsnB and AmbI3 sheds light on the possible mechanism underlying stereoselective alkene installation to complete the biosynthesis of (E)- and (Z)-3-(2-isocyanovinyl)-1H-indole 2 and 5. Establishment of a tractable isonitrile synthase system (AmbI1 and AmbI2) paves the way to elucidate the enigmatic enzyme mechanism for isocyanide formation.}, number={5}, journal={ORGANIC LETTERS}, author={Chang, Wei-Chen and Sanyal, Dev and Huang, Jhih-Liang and Ittiamornkui, Kuljira and Zhu, Qin and Liu, Xinyu}, year={2017}, month={Mar}, pages={1208–1211} }
 @article{mitchell_dipham_martinie_bergman_pollock_hu_allen_chang_silakov_bollinger_et al._2017, title={Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase}, volume={139}, ISSN={["0002-7863"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85032628383&partnerID=MN8TOARS}, DOI={10.1021/jacs.7b07374}, abstractNote={Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the l-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.}, number={39}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Mitchell, Andrew J. and Dipham, Noah P. and Martinie, Ryan J. and Bergman, Jonathan A. and Pollock, Christopher J. and Hu, Kai and Allen, Benjamin D. and Chang, Wei-chen and Silakov, Alexey and Bollinger, J. Martin, Jr. and et al.}, year={2017}, month={Oct}, pages={13830–13836} }
 @article{chang_li_lee_cronican_guo_2016, title={Mechanistic Investigation of a Non-Heme Iron Enzyme Catalyzed Epoxidation in (-)-4 '-Methoxycyclopenin Biosynthesis}, volume={138}, ISSN={["0002-7863"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84983661953&partnerID=MN8TOARS}, DOI={10.1021/jacs.6b05400}, abstractNote={Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (-)-4'-methoxycyclopenin formation uses a high-spin Fe(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on (16)O/(18)O isotope incorporation from the reactions using the native substrate, 4'-methoxydehydrocyclopeptin, and a mechanistic probe, dehydrocyclopeptin, reveals evidence supporting oxo↔hydroxo tautomerism of the Fe(IV)-oxo species in the non-heme iron enzyme catalysis.}, number={33}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Chang, Wei-chen and Li, Jikun and Lee, Justin L. and Cronican, Andrea A. and Guo, Yisong}, year={2016}, month={Aug}, pages={10390–10393} }
 @article{tamanaha_zhang_guo_chang_barr_xing_st. clair_ye_neese_bollinger_et al._2016, title={Spectroscopic Evidence for the Two C–H-Cleaving Intermediates of Aspergillus nidulans Isopenicillin N Synthase}, volume={138}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JACS.6B04065}, DOI={10.1021/jacs.6b04065}, abstractNote={The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide l-δ-aminoadipoyl-l-Cys-d-Val (ACV) on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C-H bond of the substrate: the pro-S-CCys,β-H bond for closure of the β-lactam ring, and the CVal,β-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as cosubstrate to cleave these C-H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C-H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C-H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin's founding work.}, number={28}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Tamanaha, Esta and Zhang, Bo and Guo, Yisong and Chang, Wei-chen and Barr, Eric W. and Xing, Gang and St. Clair, Jennifer and Ye, Shengfa and Neese, Frank and Bollinger, J. Martin, Jr. and et al.}, year={2016}, month={Jul}, pages={8862–8874} }
 @article{mitchell_dunham_bergman_wang_zhu_chang_liu_boal_2017, title={Structure-Guided Reprogramming of a Hydroxylase To Halogenate Its Small Molecule Substrate}, volume={56}, ISSN={["0006-2960"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85017504662&partnerID=MN8TOARS}, DOI={10.1021/acs.biochem.6b01173}, abstractNote={Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron(II) and 2-(oxo)-glutarate (FeII/2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C-H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar N-acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of FeII/2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C-H functionalization.}, number={3}, journal={BIOCHEMISTRY}, author={Mitchell, Andrew J. and Dunham, Noah P. and Bergman, Jonathan A. and Wang, Bo and Zhu, Qin and Chang, Wei-chen and Liu, Xinyu and Boal, Amie K.}, year={2017}, month={Jan}, pages={441–444} }
 @article{boal_bollinger_chang_2015, title={Assembly of the unusual oxacycles in the orthosomycin antibiotics}, volume={112}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.1514689112}, DOI={10.1073/pnas.1514689112}, abstractNote={Bacteria, fungi, and plants produce an arsenal of complex biomolecules through which they interact and compete with neighbor organisms (1). The machinery that builds these molecules is replete with iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases, enzymes that catalyze hydroxylation, halogenation, desaturation, ring-closure, ring-expansion, and stereoinversion reactions on pathways to important natural-product drugs (2). In PNAS, McCulloch, et al. provide evidence for two new reaction types by members of this versatile enzyme class (3). The reactions would explain how two unusual structures (Fig. 1A), orthoester (Fig. 1A, red) and dioxymethylene (Fig. 1A, blue) oxacycles, are installed into the oligosaccharide antibiotics known as orthosomycins (4). Reminiscent of previously demonstrated “oxacyclization” reactions in biosyntheses of such important drugs as clavulanic acid and scopolamine (2), these reactions add to the impressive repertoire of transformations by Fe/2OG enzymes and extend the intriguing mechanistic conundrum posed by the known oxacyclizations.}, number={39}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Boal, Amie K. and Bollinger, J. Martin, Jr. and Chang, Wei-chen}, year={2015}, month={Sep}, pages={11989–11990} }
 @article{martinie_livada_chang_green_krebs_bollinger_silakov_2015, title={Experimental correlation of substrate position with reaction outcome in the aliphatic halogenase, SyrB2}, volume={137}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84930670202&partnerID=MN8TOARS}, DOI={10.1021/jacs.5b03370}, abstractNote={The iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations, but how individual members of the enzyme family direct different outcomes is poorly understood. The Fe/2OG halogenase, SyrB2, chlorinates C4 of its native substrate, l-threonine appended to the carrier protein, SyrB1, but hydroxylates C5 of l-norvaline and, to a lesser extent, C4 of l-aminobutyric acid when SyrB1 presents these non-native amino acids. To test the hypothesis that positioning of the targeted carbon dictates the outcome, we defined the positions of these three substrates by measuring hyperfine couplings between substrate deuterium atoms and the stable, EPR-active iron-nitrosyl adduct, a surrogate for reaction intermediates. The Fe-(2)H distances and N-Fe-(2)H angles, which vary from 4.2 Å and 85° for threonine to 3.4 Å and 65° for norvaline, rationalize the trends in reactivity. This experimental correlation of position to outcome should aid in judging from structural data on other Fe/2OG enzymes whether they suppress hydroxylation or form hydroxylated intermediates on the pathways to other outcomes.}, number={21}, journal={Journal of the American Chemical Society}, author={Martinie, R.J. and Livada, J. and Chang, W.-C. and Green, M.T. and Krebs, C. and Bollinger, J.M. and Silakov, A.}, year={2015}, pages={6912–6919} }
 @inbook{bollinger_chang_matthews_martinie_boal_krebs_2015, title={Mechanisms of 2-oxoglutarate-dependent oxygenases: The hydroxylation paradigm and beyond}, volume={2015-January}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84950301767&partnerID=MN8TOARS}, DOI={10.1039/9781782621959-00095}, abstractNote={In humans, Fe(ii)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases are generally of the dioxygenase subclass and mediate hydroxylation of unactivated aliphatic carbon centres. Plants and microbes also employ Fe/2OG hydroxylases and, through investigations of the microbial enzymes, the mechanism of hydroxylation has been established to proceed via a potent high-spin (S = 2) Fe(iv)–oxo (ferryl) complex, which abstracts a hydrogen atom (H˙) from the substrate. Bacteria have further co-opted this central ferryl intermediate for a remarkable array of divergent reactivities, including olefin epoxidations, aliphatic halogenations, olefin-installing 1,2-dehydrogenations, oxacycle-installing 1,3- and 1,5-dehydrogenations, and a redox-neutral stereoinversion. An understanding of the mechanisms leading to this manifold of transformations, and the means by which the individual enzymes direct them, has potential to guide the design of new chemical catalysts and the development of novel bacterially- or chemo-enzymatically-derived drug compounds. In this chapter, we first summarize our understanding of hydroxylation reactions mediated by Fe/2OG hydroxylases and then review recent advances in the elucidation of two of the ‘alternative’ reactivities (halogenation and stereoinversion). Finally, we discuss the remaining, less well understood dehydrogenation reactions, highlighting possible problems with published mechanistic proposals, presenting alternatives to these published mechanisms, and briefly outlining experiments by which the operant mechanisms might be established.}, number={3}, booktitle={RSC Metallobiology}, author={Bollinger, J.M. and Chang, W.-C. and Matthews, M.L. and Martinie, R.J. and Boal, A.K. and Krebs, C.}, year={2015}, pages={95–122} }
 @article{rajakovich_nørgaard_warui_chang_li_booker_krebs_bollinger_pandelia_2015, title={Rapid Reduction of the Diferric-Peroxyhemiacetal Intermediate in Aldehyde-Deformylating Oxygenase by a Cyanobacterial Ferredoxin: Evidence for a Free-Radical Mechanism}, volume={137}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JACS.5B06345}, DOI={10.1021/jacs.5b06345}, abstractNote={Aldehyde-deformylating oxygenase (ADO) is a ferritin-like nonheme-diiron enzyme that catalyzes the last step in a pathway through which fatty acids are converted into hydrocarbons in cyanobacteria. ADO catalyzes conversion of a fatty aldehyde to the corresponding alk(a/e)ne and formate, consuming four electrons and one molecule of O2 per turnover and incorporating one atom from O2 into the formate coproduct. The source of the reducing equivalents in vivo has not been definitively established, but a cyanobacterial [2Fe-2S] ferredoxin (PetF), reduced by ferredoxin-NADP(+) reductase (FNR) using NADPH, has been implicated. We show that both the diferric form of Nostoc punctiforme ADO and its (putative) diferric-peroxyhemiacetal intermediate are reduced much more rapidly by Synechocystis sp. PCC6803 PetF than by the previously employed chemical reductant, 1-methoxy-5-methylphenazinium methyl sulfate. The yield of formate and alkane per reduced PetF approaches its theoretical upper limit when reduction of the intermediate is carried out in the presence of FNR. Reduction of the intermediate by either system leads to accumulation of a substrate-derived peroxyl radical as a result of off-pathway trapping of the C2-alkyl radical intermediate by excess O2, which consequently diminishes the yield of the hydrocarbon product. A sulfinyl radical located on residue Cys71 also accumulates with short-chain aldehydes. The detection of these radicals under turnover conditions provides the most direct evidence to date for a free-radical mechanism. Additionally, our results expose an inefficiency of the enzyme in processing its radical intermediate, presenting a target for optimization of bioprocesses exploiting this hydrocarbon-production pathway.}, number={36}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Rajakovich, Lauren J. and Nørgaard, Hanne and Warui, Douglas M. and Chang, Wei-chen and Li, Ning and Booker, Squire J. and Krebs, Carsten and Bollinger, J. Martin, Jr. and Pandelia, Maria-Eirini}, year={2015}, month={Sep}, pages={11695–11709} }
 @article{matthews_chang_layne_miles_krebs_bollinger_2014, title={Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase}, volume={10}, ISSN={1552-4450 1552-4469}, url={http://dx.doi.org/10.1038/NCHEMBIO.1438}, DOI={10.1038/nchembio.1438}, abstractNote={Iron-dependent halogenases employ cis-halo-Fe(IV)-oxo (haloferryl) complexes to functionalize unactivated aliphatic carbon centers, a capability elusive to synthetic chemists. Halogenation requires (i) coordination of a halide anion (Cl(-) or Br(-)) to the enzyme's Fe(II) cofactor, (ii) coupled activation of O2 and decarboxylation of α-ketoglutarate to generate the haloferryl intermediate, (iii) abstraction of hydrogen (H•) from the substrate by the ferryl and (iv) transfer of the cis halogen as Cl• or Br• to the substrate radical. This enzymatic solution to an unsolved chemical challenge is potentially generalizable to installation of other functional groups, provided that the corresponding anions can support the four requisite steps. We show here that the wild-type halogenase SyrB2 can indeed direct aliphatic nitration and azidation reactions by the same chemical logic. The discovery and enhancement by mutagenesis of these previously unknown reaction types suggest unrecognized or untapped versatility in ferryl-mediated enzymatic C-H bond activation.}, number={3}, journal={Nature Chemical Biology}, publisher={Springer Science and Business Media LLC}, author={Matthews, Megan L and Chang, Wei-chen and Layne, Andrew P and Miles, Linde A and Krebs, Carsten and Bollinger, J Martin, Jr}, year={2014}, month={Jan}, pages={209–215} }
 @article{mechanism of the c5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics_2014, volume={343}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84896781761&partnerID=MN8TOARS}, DOI={10.1126/science.1248000}, abstractNote={Carbapenems Through the Looking Glass
          
            The carbapenem class of antibiotics is a critical weapon in the ongoing fight against drug-resistant bacteria. Microbial biosynthesis of these compounds, which contain a strained β-lactam ring motif, proceeds via a precursor that has the wrong configuration at one of the ring carbons.
            
              Chang
              et al.
            
            (p.
            1140
            ) combined x-ray crystallography with multiple spectroscopic probes to map out the mechanism by which the CarC enzyme inverts the precursor configuration to its mirror image.
          }, number={6175}, journal={Science}, year={2014}, pages={1140–1144} }
 @article{huang_chang_lin_romo_pai_russell_russell_liu_2014, title={Mechanistic Consequences of Chiral Radical Clock Probes: Analysis of the Mononuclear Non-Heme Iron Enzyme HppE with 2-Hydroxy-3-methylenecyclopropyl Radical Clock Substrates}, volume={136}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JA4100035}, DOI={10.1021/ja4100035}, abstractNote={(S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C–H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.}, number={8}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Huang, Hui and Chang, Wei-Chen and Lin, Geng-Min and Romo, Anthony and Pai, Pei-Jing and Russell, William K. and Russell, David H. and Liu, Hung-Wen}, year={2014}, month={Feb}, pages={2944–2947} }
 @article{chang_song_liu_liu_2013, title={Current development in isoprenoid precursor biosynthesis and regulation}, volume={17}, ISSN={1367-5931}, url={http://dx.doi.org/10.1016/J.CBPA.2013.06.020}, DOI={10.1016/j.cbpa.2013.06.020}, abstractNote={Isoprenoids are one of the largest classes of natural products and all of them are constructed from two precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). For decades, the mevalonic acid (MVA) pathway was proposed to be the only IPP and DMAPP biosynthetic pathway. This review summarizes the newly discovered IPP and DMAPP production pathways since late 1990s, their distribution among different kingdoms, and their roles in secondary metabolite production. These new IPP and DMAPP production pathways include the methylerythritol phosphate (MEP) pathway, a modified MVA pathway, and the 5-methylthioadenosine shunt pathway. Relative to the studies on the MVA pathway, information on the MEP pathway regulation is limited and the mechanistic details of several of its novel transformations remain to be addressed. Current status on both MEP pathway regulation and mechanistic issues is also presented.}, number={4}, journal={Current Opinion in Chemical Biology}, publisher={Elsevier BV}, author={Chang, Wei-chen and Song, Heng and Liu, Hung-wen and Liu, Pinghua}, year={2013}, month={Aug}, pages={571–579} }
 @article{evidence that the fosfomycin-producing epoxidase, hppe, is a non-heme-iron peroxidase_2013, volume={342}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84888026754&partnerID=MN8TOARS}, DOI={10.1126/science.1240373}, abstractNote={Just Add Peroxide
          
            The HppE enzyme uses iron to catalyze oxidation of an alcohol to an epoxide ring in the biosynthesis of the antibiotic fosfomycin. Because this process is a two-electron oxidation, it has been unclear how the enzyme reduces its presumed oxidative partner O
            2
            all the way to water. Where do the two extra electrons come from?
            
              Wang
              et al.
            
            (p.
            991
            , published 10 October; see the Perspective by
            
              Raushel
            
            ) now show that HppE is actually a peroxidase, and thus reduces H
            2
            O
            2
            , for which just two electrons are sufficient. The result expands the structural scope of iron-bearing peroxidase enzymes beyond heme motifs.
          }, number={6161}, journal={Science}, year={2013}, pages={991–995} }
 @article{chang_dey_liu_mansoorabadi_moon_zhao_drennan_liu_2013, title={Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction}, volume={496}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/NATURE11998}, DOI={10.1038/nature11998}, abstractNote={The non-haem-iron-dependent enzyme HppE catalyses the final step in the biosynthesis of fosfomycin, a broad-spectrum, clinically useful antibiotic; here it is shown that HppE can also catalyse a 1,2-phosphono-migration reaction, previously undocumented for any enzyme. The last step in the biosynthesisof fosfomycin, an antibiotic used to treat diabetic foot infections and lower urinary tract infections, is catalysed by the non-haem-iron-dependent enzyme (S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE). The reaction involves an unusual dehydrogenation reaction in which the secondary alcohol of (S)-2-HPP is converted to the epoxide ring of fosfomycin. In this manuscript, the authors show that the enzyme can also catalyse a 1,2-phosphono-migration reaction, which is a biologically unprecedented chemical transformation. This finding could pave the way for the production of novel phosphonate-containing natural products and new phosphonate derivatives. (S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-haem-iron-dependent enzyme1,2,3 responsible for the final step in the biosynthesis of the clinically useful antibiotic fosfomycin4. Enzymes of this class typically catalyse oxygenation reactions that proceed via the formation of substrate radical intermediates. By contrast, HppE catalyses an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin1,5. Here we show that HppE also catalyses a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation probably involves an intermediary carbocation, based on observations with additional substrate analogues, such as (1R)-1-hydroxyl-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyse distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-haem-iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of new phosphonate derivatives.}, number={7443}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Chang, Wei-chen and Dey, Mishtu and Liu, Pinghua and Mansoorabadi, Steven O. and Moon, Sung-Ju and Zhao, Zongbao K. and Drennan, Catherine L. and Liu, Hung-wen}, year={2013}, month={Apr}, pages={114–118} }
 @book{zhao_chang_xiao_liu_liu_2013, title={Methylerythritol phosphate pathway of isoprenoid biosynthesis}, volume={82}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84878950920&partnerID=MN8TOARS}, DOI={10.1146/annurev-biochem-052010-100934}, abstractNote={ Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway–based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations. }, journal={Annual Review of Biochemistry}, author={Zhao, L. and Chang, W.-C. and Xiao, Y. and Liu, H.-W. and Liu, P.}, year={2013}, pages={497–530} }
 @article{chang_mansoorabadi_liu_2013, title={Reaction of HppE with Substrate Analogues: Evidence for Carbon–Phosphorus Bond Cleavage by a Carbocation Rearrangement}, volume={135}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JA403441X}, DOI={10.1021/ja403441x}, abstractNote={(S)-2-hydroxypropylphosphonic acid ((S)-2-HPP) epoxidase (HppE) is an unusual mononuclear non-heme iron enzyme that catalyzes the oxidative epoxidation of (S)-2-HPP in the biosynthesis of the antibiotic fosfomycin. Recently, HppE has been shown to accept (R)-1-hydroxypropylphosphonic acid as a substrate and convert it to an aldehyde product in a reaction involving a biologically unprecedented 1,2-phosphono migration. In this study, a series of substrate analogues were designed, synthesized, and used as mechanistic probes to study this novel enzymatic transformation. The resulting data, together with insights obtained from density functional theory calculations, are consistent with a mechanism of HppE-catalyzed phosphono group migration that involves the formation of a carbocation intermediate. As such, this reaction represents a new paradigm for biological C-P bond cleavage.}, number={22}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Chang, Wei-chen and Mansoorabadi, Steven O. and Liu, Hung-wen}, year={2013}, month={May}, pages={8153–8156} }
 @article{pandelia_li_nørgaard_warui_rajakovich_chang_booker_krebs_bollinger_2013, title={Substrate-Triggered Addition of Dioxygen to the Diferrous Cofactor of Aldehyde-Deformylating Oxygenase to Form a Diferric-Peroxide Intermediate}, volume={135}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/JA405047B}, DOI={10.1021/ja405047b}, abstractNote={Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or monounsaturated C(n) fatty aldehydes to formate and the corresponding C(n-1) alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 cosubstrate to the oxidation state of water and incorporates one O-atom from O2 into the formate coproduct. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2(III/III) complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2(III/III) center with resolved subsites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t(1/2) ~ 400 s at 5 °C) to produce a very modest yield of formate (<0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazinium methylsulfate ((MeO)PMS) to yield product, albeit at only ~50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or heterodinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2(III/III)-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1-C2 radical fragmentation and formation of the alk(a/e)ne and formate products.}, number={42}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Pandelia, Maria E. and Li, Ning and Nørgaard, Hanne and Warui, Douglas M. and Rajakovich, Lauren J. and Chang, Wei-chen and Booker, Squire J. and Krebs, Carsten and Bollinger, J. Martin, Jr.}, year={2013}, month={Oct}, pages={15801–15812} }
 @article{li_chang_warui_booker_krebs_bollinger_2012, title={Evidence for Only Oxygenative Cleavage of Aldehydes to Alk(a/e)nes and Formate by Cyanobacterial Aldehyde Decarbonylases}, volume={51}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi300912n}, DOI={10.1021/bi300912n}, abstractNote={Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C(n) fatty aldehydes to formate (HCO(2)(-)) and the corresponding C(n-1) alk(a/e)nes. Previous studies of the Nostoc punctiforme (Np) AD produced in Escherichia coli (Ec) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O(2) into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O(2). Two subsequent publications by Marsh and co-workers [ Das, et al. ( 2011 ) Angew. Chem. Int. Ed. 50 , 7148 - 7152 ; Eser, et al. ( 2011 ) Biochemistry 50 , 10743 - 10750 ] reported that their Ec-expressed Np and Prochlorococcus marinus (Pm) AD preparations transform aldehydes to the same products more rapidly by an O(2)-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O(2) and (ii) (18)O(2) and H(2)(18)O isotope-tracer experiments with direct mass-spectrometric detection of the HCO(2)(-) product. For multiple combinations of the AD orthologue (Np and Pm), reducing system (protein-based and chemical), and substrate (n-heptanal and n-octadecanal), our preparations strictly require O(2) for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the (18)O-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O(2) in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the C1-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO).}, number={40}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Li, Ning and Chang, Wei-chen and Warui, Douglas M. and Booker, Squire J. and Krebs, Carsten and Bollinger, J. Martin, Jr.}, year={2012}, month={Sep}, pages={7908–7916} }
 @article{huang_chang_pai_romo_mansoorabadi_russell_liu_2012, title={Evidence for Radical-Mediated Catalysis by HppE: A Study Using Cyclopropyl and Methylenecyclopropyl Substrate Analogues}, volume={134}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja3078126}, DOI={10.1021/ja3078126}, abstractNote={(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that catalyzes the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid ((S)-HPP) in the biosynthesis of the antibiotic fosfomycin. HppE also recognizes (R)-2-hydroxypropylphosphonic acid ((R)-HPP) as a substrate and converts it to 2-oxo-propylphosphonic acid. To probe the mechanisms of these HppE-catalyzed oxidations, cyclopropyl- and methylenecyclopropyl-containing compounds were synthesized and studied as radical clock substrate analogues. Enzymatic assays indicated that the (S)- and (R)-isomers of the cyclopropyl-containing analogues were efficiently converted to epoxide and ketone products by HppE, respectively. In contrast, the ultrafast methylenecyclopropyl-containing probe inactivated HppE, consistent with a rapid radical-triggered ring-opening process that leads to enzyme inactivation. Taken together, these findings provide, for the first time, experimental evidence for the involvement of a C2-centered radical intermediate with a lifetime on the order of nanoseconds in the HppE-catalyzed oxidation of (R)-HPP.}, number={39}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Huang, Hui and Chang, Wei-chen and Pai, Pei-Jing and Romo, Anthony and Mansoorabadi, Steven O. and Russell, David H. and Liu, Hung-wen}, year={2012}, month={Sep}, pages={16171–16174} }
 @article{thibodeaux_chang_liu_2012, title={Enzymatic Chemistry of Cyclopropane, Epoxide, and Aziridine Biosynthesis}, volume={112}, ISSN={0009-2665 1520-6890}, url={http://dx.doi.org/10.1021/cr200073d}, DOI={10.1021/cr200073d}, abstractNote={Cyclopropane, epoxide, and aziridine groups are three-membered ring structural elements found in a wide variety of natural products, some of which are depicted in Figure 1.1 The antibiotic and antitumor properties of many of these compounds, including duocarmycin (1),2 dynemicin (2),3 epothilone (3),4 mitomycin (4),5–8 and azinomycin (5)9–11 are well known. The pharmacological activities of others are more diverse. For example, pentalenolactone P (6)12,13 has antineoplastic and antiviral activities,14 scopolamine (7) has a subduing effect on the central nervous system,15 azicemicin (8) is active against Gram-negative bacteria and mycobacteria,16 and ficellomycin (9) shows inhibitory activities towards Gram-positive bacteria.17,18 
 
 
 
Figure 1 
 
Representative three membered ring-containing natural products. 
 
 
 
The inherent ring strain present in the small ring moiety is frequently responsible for the biological activities of these compounds, many of which are potent alkylation agents.1,19,20 For example, upon sequence specific binding to the minor groove of double stranded DNA, a twist around the amide bond of duocarmycin (1) activates the cyclopropyl ring towards alkylation by a suitably positioned adenine residue to give 10 as an adduct (Scheme 1A).21,22 Opening of the oxiranyl ring in the reduced dynemicin A (11) is known to trigger the rearrangement of the enediyne group to a 1,4-dehydrobenzene biradical (11→12) that initiates DNA degradation (Scheme 1B).23,24 The reductive activation of mitomycin C (4) involves opening of the aziridine ring (13→14), which unmasks the electrophilic site at C-1 and results in DNA alkylation (14→15→16, Scheme 1C).5–7 In other examples, such as the tubulin-binding cytotoxin epothilone (3, Figure 1), the epoxide ring introduces a rigid structural element into the parent compound. While derivatives lacking the epoxide ring exhibit similar activities as the parent compound,25 the epoxide moiety may be important for the directing/binding of epothilone to its biological target in the cell.4,26,27 
 
 
 
Scheme 1 
 
Mechanism of action of (A) duocarmycin, (B) dynemicin, and (C) mitomycin. 
 
 
 
Despite the recent advances in our understanding of the biosynthesis of many different types of natural products, the specific enzymes responsible for making these strained, 3-membered rings have only been identified in a few cases, and many of the mechanistic details regarding small ring closure remain obscure. Due to the impressive array of diverse biological activities exhibited by these small-ring containing compounds and their potential applications as therapeutic agents as well as mechanistic probes for studying enzyme catalysis,1,19,20,28–30 a thorough understanding of their biosynthesis is warranted. These studies are a crucial first step in maximizing the potential of these compounds as general mechanistic probes, or as specific tools in the rational design of drugs with optimal in vivo specificity. In this review, we will illustrate the established biosynthetic strategies for construction of cyclopropane, epoxide, and aziridine rings, focusing primarily on studies that were performed in the last decade. Only those pathways that have been genetically and/or biochemically verified will be discussed. Special attention will be directed to the prototypes of chemical transformations observed in small-ring biosynthesis and, when applicable, mechanistic details of the enzymes involved. In addition, we will highlight the occurrence of several poorly understood, but potentially novel modes of enzyme-catalyzed small-ring biosynthesis that merit further investigation.}, number={3}, journal={Chemical Reviews}, publisher={American Chemical Society (ACS)}, author={Thibodeaux, Christopher J. and Chang, Wei-chen and Liu, Hung-wen}, year={2012}, month={Mar}, pages={1681–1709} }
 @article{chang_xiao_liu_liu_2011, title={Mechanistic Studies of an IspH-Catalyzed Reaction: Implications for Substrate Binding and Protonation in the Biosynthesis of Isoprenoids}, volume={50}, ISSN={1433-7851}, url={http://dx.doi.org/10.1002/anie.201104124}, DOI={10.1002/anie.201104124}, abstractNote={Isoprenoids are found widely in nature and have remarkably diverse structures.[1] They are utilized by all living organisms to fulfill a variety of biological roles, including serving as structural components of cell membranes, key constituents of electron transport chains, and hormones to regulate various physiological processes.[2] Many isoprenoids, produced as secondary metabolites, function as defense agents for the producers and have been one of the rich sources for human medicines.[2–3]}, number={51}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={Chang, Wei-chen and Xiao, Youli and Liu, Hung-wen and Liu, Pinghua}, year={2011}, month={Oct}, pages={12304–12307} }
 @article{sun_ruszczycky_chang_thibodeaux_liu_2012, title={Nucleophilic Participation of Reduced Flavin Coenzyme in Mechanism of UDP-galactopyranose Mutase}, volume={287}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M111.312538}, DOI={10.1074/jbc.M111.312538}, abstractNote={Background: UDP-galactopyranose mutase (UGM) requires the reduced FAD coenzyme to interconvert UDP-galactopyranose and UDP-galactofuranose. Results: Structural perturbations of the coenzyme inhibit bond cleavage in the substrate. Conclusion: Concerted bond breaking and formation between substrate and coenzyme occur during UGM catalysis. Significance: Mechanistic understanding of UGM offers new insight for clinically relevant inhibitor design. UDP-galactopyranose mutase (UGM) requires reduced FAD (FADred) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FADred at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FADred in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FADred is unable to catalyze PIX of the bridging C1–OPβ oxygen of UDP-Galp, suggesting a direct role for the FADred N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of kcat versus the nucleophilicity of N5 of FADred gave a slope of ρ = −2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an SN2-type displacement of UDP from UDP-Galf/p by N5 of FADred.}, number={7}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Sun, He G. and Ruszczycky, Mark W. and Chang, Wei-chen and Thibodeaux, Christopher J. and Liu, Hung-wen}, year={2012}, pages={4602–4608} }
 @article{xiao_chang_liu_liu_2011, title={Study of IspH, a Key Enzyme in the Methylerythritol Phosphate Pathway Using Fluoro-Substituted Substrate Analogues}, volume={13}, ISSN={1523-7060 1523-7052}, url={http://dx.doi.org/10.1021/ol202559r}, DOI={10.1021/ol202559r}, abstractNote={IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies of IspH using fluoro-substituted substrate analogues to dissect the contributions of several factors to IspH catalysis, including the coordination of the HMBPP C(4)-OH group to the iron-sulfur cluster, the H-bonding network in the active site, and the electronic properties of the substrates, are reported.}, number={21}, journal={Organic Letters}, publisher={American Chemical Society (ACS)}, author={Xiao, Youli and Chang, Wei-chen and Liu, Hung-wen and Liu, Pinghua}, year={2011}, month={Nov}, pages={5912–5915} }
 @article{thibodeaux_chang_liu_2010, title={Linear Free Energy Relationships Demonstrate a Catalytic Role for the Flavin Mononucleotide Coenzyme of the Type II Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja104090m}, DOI={10.1021/ja104090m}, abstractNote={The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible isomerization of the two ubiquitous isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are required to initiate the biosynthesis of all isoprenoid compounds found in nature. The overall chemical transformation catalyzed by IDI-2 involves a net 1,3-proton addition/elimination reaction. Surprisingly, IDI-2 requires a reduced flavin mononucleotide (FMN) coenzyme to carry out this redox neutral isomerization. The exact function of FMN in catalysis has not yet been clearly defined. To provide mechanistic insight into the role of the reduced flavin in IDI-2 catalysis, several FMN analogues with altered electronic properties were chemoenzymatically prepared, and their effects on the kinetic properties of the IDI-2 catalyzed reaction were investigated. Linear free energy relationships (LFERs) between the electronic properties of the flavin and the steady state kinetic parameters of the IDI-2 catalyzed reaction were observed. The LFER studies are complemented with kinetic isotope effect studies and kinetic characterization of an active site mutant enzyme (Q154N). Cumulatively, the data presented in this work (and in other studies) suggest that the reduced FMN coenzyme of IDI-2 functions as an acid/base catalyst, with the N5 atom of the flavin likely playing a critical role in the deprotonation of IPP en route to DMAPP formation. Several potential chemical mechanisms involving the reduced flavin as an acid/base catalyst are presented and discussed.}, number={29}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Thibodeaux, Christopher J. and Chang, Wei-chen and Liu, Hung-wen}, year={2010}, month={Jul}, pages={9994–9996} }
 @article{thibodeaux_mansoorabadi_kittleman_chang_liu_2008, title={Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase fromStaphylococcus aureus}, volume={47}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi701467g}, DOI={10.1021/bi701467g}, abstractNote={The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steady-state and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect (D2OVmax = 1.5) was measured on vo in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity (DVmax = 1.8) and on the decay rate of the flavin intermediate (Dks = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2H]-IPP. Taken together, these data suggest that the C2-H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures.}, number={8}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Thibodeaux, Christopher J. and Mansoorabadi, Steven O. and Kittleman, William and Chang, Wei-chen and Liu, Hung-wen}, year={2008}, month={Feb}, pages={2547–2558} }
 @article{munos_moon_mansoorabadi_chang_hong_yan_liu_liu_2008, title={Purification and Characterization of the Epoxidase Catalyzing the Formation of Fosfomycin from Pseudomonas syringae}, volume={47}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi800877v}, DOI={10.1021/bi800877v}, abstractNote={The final step in the biosynthesis of fosfomycin in Streptomyces wedmorensis is catalyzed by (S)-2-hydroxypropylphosphonic acid (HPP) epoxidase (Sw-HppE). A homologous enzyme from Pseudomonas syringae whose encoding gene (orf3) shares a relatively low degree of sequence homology with the corresponding Sw-HppE gene has recently been isolated. This purified P. syringae protein was determined to catalyze the epoxidation of (S)-HPP to fosfomycin and the oxidation of (R)-HPP to 2-oxopropylphosphonic acid under the same conditions as Sw-HppE. Therefore, this protein is indeed a true HPP epoxidase and is termed Ps-HppE. Like Sw-HppE, Ps-HppE was determined to be post-translationally modified by the hydroxylation of a putative active site tyrosine (Tyr95). Analysis of the Fe(II) center by EPR spectroscopy using NO as a spin probe and molecular oxygen surrogate reveals that Ps-HppE's metal center is similar, but not identical, to that of Sw-HppE. The identity of the rate-determining step for the (S)-HPP and (R)-HPP reactions was determined by measuring primary deuterium kinetic effects, and the outcome of these results was correlated with density functional theory calculations. Interestingly, the reaction using the nonphysiological substrate (R)-HPP was 1.9 times faster than that with (S)-HPP for both Ps-HppE and Sw-HppE. This is likely due to the difference in bond dissociation energy of the abstracted hydrogen atom for each respective reaction. Thus, despite the low level of amino acid sequence identity, Ps-HppE is a close mimic of Sw-HppE, representing a second example of a non-heme iron-dependent enzyme capable of catalyzing dehydrogenation of a secondary alcohol to form a new C−O bond.}, number={33}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Munos, Jeffrey W. and Moon, Sung-Ju and Mansoorabadi, Steven O. and Chang, Weichen and Hong, Lin and Yan, Feng and Liu, Aimin and Liu, Hung-wen}, year={2008}, month={Aug}, pages={8726–8735} }