@misc{zimmer_barycki_simpson_2022, title={Mechanisms of coordinating hyaluronan and glycosaminoglycan production by nucleotide sugars}, volume={322}, ISSN={["1522-1563"]}, DOI={10.1152/ajpcell.00130.2022}, abstractNote={ Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it. However, the nucleotide-activated sugar substrates for hyaluronan synthesis by HAS also participate in exquisitely fine-tuned cross-talking pathways that intersect with glycosaminoglycan production and central carbohydrate metabolism. Multiple UDP-sugars have alternative metabolic fates and exhibit coordinated and reciprocal allosteric control of enzymes within their biosynthetic pathways to preserve appropriate precursor ratios for accurate partitioning among downstream products, while also sensing and maintaining energy homeostasis. Since the dysregulation of nucleotide sugar and hyaluronan synthesis is associated with multiple pathologies, these pathways offer opportunities for therapeutic intervention. Recent structures of several key rate-limiting enzymes in the UDP-sugar synthesis pathways have offered new insights to the overall regulation of hyaluronan production by precursor fate decisions. The details of UDP-sugar control and the structural basis for underlying mechanisms are discussed in this review. }, number={6}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY}, author={Zimmer, Brenna M. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2022}, month={Jun}, pages={C1201–C1213} }
 @misc{zimmer_barycki_simpson_2021, title={Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase}, volume={69}, ISSN={["1551-5044"]}, DOI={10.1369/0022155420947500}, abstractNote={Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:}, number={1}, journal={JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY}, author={Zimmer, Brenna M. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2021}, month={Jan}, pages={13–23} }
 @article{hengel_bosso-lefevre_grady_szenker-ravi_li_pierce_lebigot_tan_eio_narayanan_et al._2020, title={Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy}, volume={11}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-020-14360-7}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, author={Hengel, Holger and Bosso-Lefevre, Celia and Grady, George and Szenker-Ravi, Emmanuelle and Li, Hankun and Pierce, Sarah and Lebigot, Elise and Tan, Thong-Teck and Eio, Michelle Y. and Narayanan, Gunaseelan and et al.}, year={2020}, month={Jan} }
 @article{barycki_2017, title={Covering their bases: The phosphobase methylation pathway in plants}, volume={292}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.h117.000712}, abstractNote={Phosphoethanolamine methyltransferases add three methyl groups successively to their substrate to produce phosphocholine, an important precursor for phospholipid biosynthesis in diverse organisms. New work from Lee and Jez reveals critical domain movements that explain how multiple methylation reactions are uniquely coordinated by plant methyltransferases and provides insights into the evolution of this class of enzymes. As opposed to closely related family members, the intermediates in this pathway are likely shuttled between two tethered domains to ensure complete methylation.}, number={52}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Barycki, Joseph J.}, year={2017}, month={Dec}, pages={21703–21704} }
 @article{grady_thelen_albers_ju_guo_barycki_simpson_2016, title={Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking}, volume={55}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/ACS.BIOCHEM.6B00259}, DOI={10.1021/ACS.BIOCHEM.6B00259}, abstractNote={The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.}, number={22}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Grady, George and Thelen, Ashley and Albers, Jaleen and Ju, Tong and Guo, Jiantao and Barycki, Joseph J. and Simpson, Melanie A.}, year={2016}, month={May}, pages={3157–3164} }
 @article{zimmer_howell_wei_ma_romsdahl_loughman_markham_seravalli_barycki_simpson_2016, title={Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential}, volume={7}, ISSN={1868-8497 1868-8500}, url={http://dx.doi.org/10.1007/S12672-016-0268-Z}, DOI={10.1007/S12672-016-0268-Z}, abstractNote={Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD(+)-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen-dependent versus castration-resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration-resistant tumor cells expressed higher levels of UGDH, UGT2B15, and UGT2B17. However, the magnitude of androgen-activated UGDH and prostate-specific antigen (PSA) expression, as well as the androgen receptor (AR)-dependent repression of UGT2B15 and UGT2B17, was blunted several-fold in these cells. Consistent with these results, the ligand-activated binding of AR to the PSA promoter and subsequent transcriptional activation were also significantly reduced in castration-resistant cells. Analysis of the UDP-sugar pools and flux through pathways downstream of UDP-glucuronate production revealed that these glucuronidation precursor metabolites were channeled through proteoglycan and glycosaminoglycan biosynthetic pathways, leading to increased surface expression of Notch1. Knockdown of UGDH diminished Notch1 and increased glucuronide output. Overall, these results support a model in which the aberrant partitioning of UDP-glucuronate and other UDP-sugars into alternative pathways during androgen deprivation contributes to the loss of prostate tumor cell androgen sensitivity by promoting altered cell surface proteoglycan expression.}, number={4}, journal={Hormones and Cancer}, publisher={Springer Nature}, author={Zimmer, Brenna M . and Howell, Michelle E. and Wei, Qin and Ma, Linlin and Romsdahl, Trevor and Loughman, Eileen G. and Markham, Jonathan E. and Seravalli, Javier and Barycki, Joseph J. and Simpson, Melanie A.}, year={2016}, month={Jun}, pages={260–271} }
 @article{wu_kim_seravalli_barycki_hart_gohara_di cera_jung_kosman_lee_2016, title={Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase}, volume={291}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M115.700500}, DOI={10.1074/JBC.M115.700500}, abstractNote={Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K+) compartmentalization to the trans-Golgi network via Kha1p, a K+/H+ exchanger. K+ in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K+ is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K+ acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K+ compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K+ in the binding of copper to apoFet3p and identifies a K+/H+ exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast.}, number={18}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Wu, Xiaobin and Kim, Heejeong and Seravalli, Javier and Barycki, Joseph J. and Hart, P. John and Gohara, David W. and Di Cera, Enrico and Jung, Won Hee and Kosman, Daniel J. and Lee, Jaekwon}, year={2016}, month={Mar}, pages={9796–9806} }
 @article{ozer_dlouhy_thornton_hu_liu_barycki_balk_outten_2015, title={Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System}, volume={290}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M115.682179}, DOI={10.1074/JBC.M115.682179}, abstractNote={Background: The mitochondrial sulfhydryl oxidase Erv1 is implicated in cytosolic iron-sulfur protein maturation and iron regulation. Results: An erv1 yeast strain used in previous iron metabolism studies is glutathione-deficient, and erv1/mia40 mutants with sufficient glutathione do not exhibit iron-related defects. Conclusion: Iron homeostasis is independent of Erv1/Mia40 function. Significance: The unexplained role of Erv1 in yeast iron metabolism is resolved. The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.}, number={46}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Ozer, Hatice K. and Dlouhy, Adrienne C. and Thornton, Jeremy D. and Hu, Jingjing and Liu, Yilin and Barycki, Joseph J. and Balk, Janneke and Outten, Caryn E.}, year={2015}, month={Sep}, pages={27829–27840} }
 @article{mcatee_berkebile_elowsky_fangman_barycki_wahl_khalimonchuk_naslavsky_caplan_simpson_2015, title={Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking}, volume={290}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M115.647446}, DOI={10.1074/JBC.M115.647446}, abstractNote={Background: Hyal1 is a turnover enzyme for hyaluronan that accelerates metastatic cancer by increasing cell motility. Results: Hyal1-overexpressing cells have a higher rate of endocytosis that impacts cargo internalization and recycling. Conclusion: The higher rate of vesicle trafficking increases motility receptor function and nutrient uptake. Significance: This novel mechanism implicates Hyal1 trafficking in multiple signaling events during tumor progression. Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific colocalization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found that enzymatic activity of Hyal1 was necessary for normal localization within the cell as Hyal1-E131Q was mainly detected within the endoplasmic reticulum. Expression of a HA-binding point mutant, Hyal1-Y202F, revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space are critical for rapid HA internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, promoting aggressive tumor cell behavior.}, number={21}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={McAtee, Caitlin O. and Berkebile, Abigail R. and Elowsky, Christian G. and Fangman, Teresa and Barycki, Joseph J. and Wahl, James K., III and Khalimonchuk, Oleh and Naslavsky, Naava and Caplan, Steve and Simpson, Melanie A.}, year={2015}, month={Apr}, pages={13144–13156} }
 @article{hyde_thelen_barycki_simpson_2013, title={UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces}, volume={288}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M113.519090}, DOI={10.1074/JBC.M113.519090}, abstractNote={Background: UDP-glucose dehydrogenase (UGDH) mutants were engineered to perturb hexamer:dimer quaternary structure equilibrium. Results: Dimeric species of UGDH have reduced activity in vitro and in supporting hyaluronan production by cultured cells. Conclusion: Only dynamic UGDH hexamers support robust cellular function. Significance: Manipulation of UGDH activity by hexamer stabilization may offer new therapeutic options in cancer and other pathologies. UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.}, number={49}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Hyde, Annastasia S. and Thelen, Ashley M. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2013}, month={Oct}, pages={35049–35057} }
 @article{hyde_farmer_easley_van lammeren_christoffels_barycki_bakkers_simpson_2012, title={UDP-glucose Dehydrogenase Polymorphisms from Patients with Congenital Heart Valve Defects Disrupt Enzyme Stability and Quaternary Assembly}, volume={287}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M112.395202}, DOI={10.1074/jbc.M112.395202}, abstractNote={Background: UDP-glucose dehydrogenase (UGDH) polymorphisms were identified in a screen of candidate genes for heart valve defects. Results: Two individual mutants fail to rescue cardiac valve defects in UGDH-deleted zebrafish and have reduced stability in vitro. Conclusion: UGDH loss of function mutations result in a subset of human congenital cardiac valve defects caused by reduced enzyme activity during morphogenesis. Significance: Screening these alleles could predict valve defects. Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects.}, number={39}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Hyde, Annastasia S. and Farmer, Erin L. and Easley, Katherine E. and van Lammeren, Kristy and Christoffels, Vincent M. and Barycki, Joseph J. and Bakkers, Jeroen and Simpson, Melanie A.}, year={2012}, month={Jul}, pages={32708–32716} }
 @article{willis_liu_biterova_simpson_kim_lee_barycki_2011, title={Enzymatic Defects Underlying Hereditary Glutamate Cysteine Ligase Deficiency Are Mitigated by Association of the Catalytic and Regulatory Subunits}, volume={50}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi200708w}, DOI={10.1021/bi200708w}, abstractNote={Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.}, number={29}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Willis, Melanie Neely and Liu, Yilin and Biterova, Ekaterina I. and Simpson, Melanie A. and Kim, Heejeong and Lee, Jaekwon and Barycki, Joseph J.}, year={2011}, month={Jul}, pages={6508–6517} }
 @article{biterova_barycki_2010, title={Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase}, volume={285}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M110.104802}, DOI={10.1074/jbc.M110.104802}, abstractNote={Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 Å; R = 19.9%, Rfree = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg2+ coordination in the ATP-binding site. l-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 Å; R = 18.1%, Rfree = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.}, number={19}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Biterova, Ekaterina I. and Barycki, Joseph J.}, year={2010}, month={Mar}, pages={14459–14466} }
 @article{williams_cullati_sand_biterova_barycki_2009, title={Crystal Structure of Acivicin-Inhibited γ-Glutamyltranspeptidase Reveals Critical Roles for Its C-Terminus in Autoprocessing and Catalysis†‡}, volume={48}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi8014955}, DOI={10.1021/bi8014955}, abstractNote={Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a general gamma-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used gamma-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k(max) = 0.033 +/- 0.006 s(-1) and K(I) = 19.7 +/- 7.2 microM. Structure determination of acivicin-modified HpGT (1.7 A; R(factor) = 17.9%; R(free) = 20.8%) demonstrates that acivicin is accommodated within the gamma-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.}, number={11}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Williams, Kristin and Cullati, Sierra and Sand, Aaron and Biterova, Ekaterina I. and Barycki, Joseph J.}, year={2009}, month={Mar}, pages={2459–2467} }
 @article{zhang_bharadwaj_casper_barkley_barycki_simpson_2009, title={Hyaluronidase Activity of Human Hyal1 Requires Active Site Acidic and Tyrosine Residues}, volume={284}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M900210200}, DOI={10.1074/jbc.M900210200}, abstractNote={Hyaluronidases are a family of endolytic glycoside hydrolases that cleave the β1-4 linkage between N-acetylglucosamine and glucuronic acid in hyaluronan polymers via a substrate-assisted mechanism. In humans, turnover of hyaluronan by this enzyme family is critical for normal extracellular matrix remodeling. However, elevated expression of the Hyal1 isozyme accelerates tumor growth and metastatic progression. In this study, we used structural information, site-directed mutagenesis, and steady state enzyme kinetics to probe molecular determinants of human Hyal1 function. Mutagenesis of active site residues Glu131 and Tyr247 to Gln and Phe, respectively, eliminated activity at all hyaluronan concentrations (to 125 μm or 2.5 mg/ml). Conservative mutagenesis of Asp129 and Tyr202 significantly impaired catalysis by increases of 5- and 10-fold in apparent Km and reductions in Vmax of 95 and 50%, respectively. Tyr247 and Asp129 are required for stabilization of the catalytic nucleophile, which arises as a resonance intermediate of N-acetylglucosamine on the substrate. Glu131 is a likely proton donor for the hydroxyl leaving group. Tyr202 is a substrate binding determinant. General disulfide reduction had no effect on activity in solution, but enzymatic deglycosylation reduced Hyal1 activity in a time-dependent fashion. Mutagenesis identified Asn350 glycosylation as the requisite modification. Deletion of the C-terminal epidermal growth factor-like domain, in which Asn350 is located, also eliminated activity, irrespective of glycosylation. Collectively, these studies define key components of Hyal1 active site catalysis, and structural factors critical for stability. Such detailed understanding will allow rational design of enzyme modulators.}, number={14}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Zhang, Ling and Bharadwaj, Alamelu G. and Casper, Andrew and Barkley, Joel and Barycki, Joseph J. and Simpson, Melanie A.}, year={2009}, month={Feb}, pages={9433–9442} }
 @article{biterova_barycki_2009, title={Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase}, volume={284}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M109.025114}, DOI={10.1074/jbc.M109.025114}, abstractNote={Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl2 (2.1 Å; R = 18.2%, Rfree = 21.9%), and in complex with glutamate, MgCl2, and ADP (2.7 Å; R = 19.0%, Rfree = 24.2%). Inspection of these structures reveals an unusual binding pocket for the α-carboxylate of the glutamate substrate and an ATP-independent Mg2+ coordination site, clarifying the Mg2+ dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.}, number={47}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Biterova, Ekaterina I. and Barycki, Joseph J.}, year={2009}, month={Sep}, pages={32700–32708} }
 @article{morrow_williams_sand_boanca_barycki_2007, title={Characterization of Helicobacter pyloriγ-Glutamyltranspeptidase Reveals the Molecular Basis for Substrate Specificity and a Critical Role for the Tyrosine 433-Containing Loop in Catalysis†,‡}, volume={46}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi701599e}, DOI={10.1021/bi701599e}, abstractNote={Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a member of the N-terminal nucleophile hydrolase superfamily. It is translated as an inactive 60 kDa polypeptide precursor that undergoes intramolecular autocatalytic cleavage to generate a fully active heterodimer composed of a 40 kDa and a 20 kDa subunit. The resultant N-terminus, Thr 380, has been shown to be the catalytic nucleophile in both autoprocessing and enzymatic reactions. Once processed, HpGT catalyzes the hydrolysis of the gamma-glutamyl bond in glutathione and its conjugates. To facilitate the determination of physiologically relevant substrates for the enzyme, crystal structures of HpGT in complex with glutamate (1.6 A, Rfactor = 16.7%, Rfree = 19.0%) and an inactive HpGT mutant, T380A, in complex with S-(nitrobenzyl)glutathione (1.55 A, Rfactor = 18.7%, Rfree = 21.8%) have been determined. Residues that comprise the gamma-glutamyl binding site are primarily located in the 20 kDa subunit and make numerous hydrogen bonds with the alpha-amino and alpha-carboxylate groups of the substrate. In contrast, a single hydrogen bond occurs between the T380A mutant and the remainder of the ligand. Lack of specific coordination beyond the gamma-glutamyl moiety may account for the substrate binding permissiveness of the enzyme. Structural analysis was combined with site-directed mutagenesis of residues involved in maintaining the conformation of a loop region that covers the gamma-glutamyl binding site. Results provide evidence that access to this buried site may occur through conformational changes in the Tyr 433-containing loop, as disruption of the intricate hydrogen-bond network responsible for optimal placement of Tyr 433 significantly diminishes catalytic activity.}, number={46}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Morrow, Amy L. and Williams, Kristin and Sand, Aaron and Boanca, Gina and Barycki, Joseph J.}, year={2007}, month={Nov}, pages={13407–13414} }
 @article{easley_sommer_boanca_barycki_simpson_2007, title={Characterization of Human UDP-Glucose Dehydrogenase Reveals Critical Catalytic Roles for Lysine 220 and Aspartate 280†}, volume={46}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi061537d}, DOI={10.1021/bi061537d}, abstractNote={Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.}, number={2}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Easley, Katherine E. and Sommer, Brandi J. and Boanca, Gina and Barycki, Joseph J. and Simpson, Melanie A.}, year={2007}, month={Jan}, pages={369–378} }
 @article{boanca_sand_barycki_2006, title={Uncoupling the Enzymatic and Autoprocessing Activities of Helicobacter pylori γ-Glutamyltranspeptidase}, volume={281}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M603381200}, DOI={10.1074/jbc.M603381200}, abstractNote={γ-Glutamyltranspeptidase (γGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the γ-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori γ-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported γGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the γ-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian γGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the γ-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.}, number={28}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Boanca, Gina and Sand, Aaron and Barycki, Joseph J.}, year={2006}, month={May}, pages={19029–19037} }
 @article{biterova_turanov_gladyshev_barycki_2005, title={Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism}, volume={102}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0504218102}, DOI={10.1073/pnas.0504218102}, abstractNote={Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 Å;R= 22.2%;Rfree= 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin–thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 Å;R= 24.1%;Rfree= 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.}, number={42}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Biterova, E. I. and Turanov, A. A. and Gladyshev, V. N. and Barycki, J. J.}, year={2005}, month={Oct}, pages={15018–15023} }
 @article{sommer_barycki_simpson_2004, title={Characterization of Human UDP-glucose Dehydrogenase}, volume={279}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M401928200}, DOI={10.1074/jbc.M401928200}, abstractNote={UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for Km and Vmax that agree well with those reported for a bovine homolog. We used crystal coordinates for Streptococcus pyogenes UGDH in complex with NAD+ cofactor and UDP-glucose substrate to generate a model of the enzyme active site. Based on this model, we selected Cys-276 and Lys-279 as likely catalytic residues and converted them to serine and alanine, respectively. Enzymatic activity of C276S and K279A point mutants was not measurable under normal assay conditions. Rate constants measured over several hours demonstrated that K279A continued to turn over, although 250-fold more slowly than wild type enzyme. C276S, however, performed only a single round of oxidation, indicating that it is essential for the second oxidation. This result is consistent with the postulated role of Cys-276 as a catalytic residue and supports its position in the reaction mechanism for the human enzyme. Lys-279 is likely to have a role in positioning active site residues and in maintaining the hexameric quaternary structure.}, number={22}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Sommer, Brandi J. and Barycki, Joseph J. and Simpson, Melanie A.}, year={2004}, month={Mar}, pages={23590–23596} }