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Structure and Mechanism of Human UDP-glucose 6-Dehydrogenase*

  • Sigrid Egger
    Affiliations
    Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria
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  • Apirat Chaikuad
    Affiliations
    Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, United Kingdom
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  • Kathryn L. Kavanagh
    Affiliations
    Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, United Kingdom
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  • Udo Oppermann
    Affiliations
    Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, United Kingdom

    Botnar Research Centre, NIHR Oxford Biomedical Research Unit, University of Oxford, Oxford OX3 7LD, United Kingdom
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  • Bernd Nidetzky
    Correspondence
    To whom correspondence should be addressed. Tel.: 43-316-873-8400
    Affiliations
    Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria
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  • Author Footnotes
    * This work was supported by the Austrian Science Fund DK Molecular Enzymology W901-B05 and the Structural Genomics Consortium registered charity (number 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, the Canadian Foundation for Innovation, Vinnova, the Swedish Strategic Research Foundation, the Knut and Alice Wallenberg Foundation, and the Karolinska Institutet. This work was additionally supported by the NIHR Oxford Biomedical Research Unit.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental “Materials and Methods,” Figs. S1 and S2, and additional references.
Open AccessPublished:April 18, 2011DOI:https://doi.org/10.1074/jbc.M111.234682
      Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD+-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD+. The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD+ binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr14, Glu161, and Glu165 participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD+ binding domain.

      Introduction

      UDP-glucuronic acid (UDP-GlcUA)
      The abbreviations used are: UDP-GlcUA
      UDP-glucuronic acid
      PDB
      Protein Data Bank
      h
      human
      UGDH
      UDP-glucose 6-dehydrogenase
      Bistris propane
      1,3-bis[tris(hydroxymethyl)methylamino]propane
      ITC
      isothermal titration calorimetry
      UDP-Glc
      UDP-glucose.
      fulfills a range of vitally important functions in human physiology. It is a precursor for synthesis of extracellular matrix glycosaminoglycans (heparin, hyaluronan, and chondroitin sulfate), which in turn play significant roles in diverse cellular processes like signaling, wound healing, inflammation, morphogenesis, and matrix organization as well as in the pathobiology of cancer (
      • Bharadwaj A.G.
      • Rector K.
      • Simpson M.A.
      ,
      • Lin S.L.
      • Chang D.
      • Ying S.Y.
      ,
      • Slomiany M.G.
      • Toole B.P.
      ,
      • Tammi R.H.
      • Kultti A.
      • Kosma V.M.
      • Pirinen R.
      • Auvinen P.
      • Tammi M.I.
      ,
      • Toole B.P.
      • Slomiany M.G.
      ,
      • Varki A.
      • Cummings R.D.
      • Esko J.D.
      • Freeze H.H.
      • Stanley P.
      • Bertozzi C.R.
      • Hart G.W.
      • Etzler M.E.
      ). UDP-GlcUA is the substrate for enzymatic glucuronidation, a key step in numerous detoxification pathways carried out in liver and intestine. Biosynthetic routes toward various UDP-sugars involve UDP-GlcUA as the central intermediate. UDP-GlcUA is derived from UDP-glucose (UDP-Glc) whereby UDP-glucose 6-dehydrogenase (UGDH; EC 1.1.1.22) catalyzes, in two successive NAD+-dependent steps without release of the intermediate aldehyde, the oxidation of the substrate C6 alcohol into the corresponding carboxylic acid (
      • Feingold D.S.
      • Franzen J.S.
      ,
      • Ge X.
      • Campbell R.E.
      • van de Rijn I.
      • Tanner M.E.
      ,
      • Sommer B.J.
      • Barycki J.J.
      • Simpson M.A.
      ). Elevated production of hyaluronan strongly promotes epithelial cancer progression, and it was shown that limitation of hyaluronan synthesis at the level of UDP-GlcUA has significant potential in slowing tumor growth (
      • Auvinen P.
      • Tammi R.
      • Parkkinen J.
      • Tammi M.
      • Agren U.
      • Johansson R.
      • Hirvikoski P.
      • Eskelinen M.
      • Kosma V.M.
      ,
      • Huh J.W.
      • Choi M.M.
      • Yang S.J.
      • Yoon S.Y.
      • Choi S.Y.
      • Cho S.W.
      ,
      • Simpson M.A.
      • Wilson C.M.
      • McCarthy J.B.
      ,
      • Wang T.P.
      • Pan Y.R.
      • Fu C.Y.
      • Chang H.Y.
      ,
      • Wei Q.
      • Galbenus R.
      • Raza A.
      • Cerny R.L.
      • Simpson M.A.
      ). Moreover, UGDH was recently proposed as a novel biomarker for prostate cancer (
      • Huang D.
      • Casale G.P.
      • Tian J.
      • Lele S.M.
      • Pisarev V.M.
      • Simpson M.A.
      • Hemstreet 3rd, G.P.
      ). Therefore, restriction of UDP-GlcUA availability in cancer cells through inhibition of human UGDH (hUGDH) presents a clear target for therapy. Design of useful antagonists for hUGDH relies on known relationships of structure and function for this enzyme, which in the absence of high resolution structural information on hUGDH are lacking in their basis. UGDH from the bacterium Streptococcus pyogenes (SpUGDH) is the only member of the large and divergent UGDH protein family (
      • Egger S.
      • Chaikuad A.
      • Kavanagh K.L.
      • Oppermann U.
      • Nidetzky B.
      ) for which a crystal structure has so far been reported (
      • Campbell R.E.
      • Mosimann S.C.
      • van De Rijn I.
      • Tanner M.E.
      • Strynadka N.C.
      ). However, considering the low level of sequence identity (23%) between hUGDH and SpUGDH, it would be a pointless exercise trying to infer relevant properties of the human enzyme from structure-function relationship data for the bacterial ortholog (
      • Ge X.
      • Campbell R.E.
      • van de Rijn I.
      • Tanner M.E.
      ,
      • Campbell R.E.
      • Mosimann S.C.
      • van De Rijn I.
      • Tanner M.E.
      • Strynadka N.C.
      ,
      • Campbell R.E.
      • Sala R.F.
      • van de Rijn I.
      • Tanner M.E.
      ,
      • Campbell R.E.
      • Tanner M.E.
      ,
      • Campbell R.E.
      • Tanner M.E.
      ,
      • Ge X.
      • Penney L.C.
      • van de Rijn I.
      • Tanner M.E.
      ). This study reports on a comprehensive structural characterization of hUGDH.
      Four-electron enzymatic oxidation of UDP-Glc is complex and involves the consecutive activities of alcohol dehydrogenase and aldehyde dehydrogenase, both recruited from a single UGDH catalytic center. Combined evidence from previous mechanistic studies of bovine liver UGDH (
      • Feingold D.S.
      • Franzen J.S.
      ,
      • Ordman A.B.
      • Kirkwood S.
      ,
      • Schiller J.G.
      • Bowser A.M.
      • Feingold D.S.
      ,
      • Strominger J.L.
      • Kalckar H.M.
      • Axelrod J.
      • Maxwell E.S.
      ) and SpUGDH (
      • Ge X.
      • Campbell R.E.
      • van de Rijn I.
      • Tanner M.E.
      ,
      • Campbell R.E.
      • Tanner M.E.
      ,
      • Ge X.
      • Penney L.C.
      • van de Rijn I.
      • Tanner M.E.
      ) supports enzymatic reaction in three catalytic steps via thiohemiacetal and thioester intermediates, as shown in Scheme 1. The proposed mechanism necessitates that the different chemical steps of the enzymatic reaction are precisely timed with intermediate physical steps of coenzyme binding and release. A central question of UGDH catalytic function therefore is how the enzyme achieves exact orchestration of these multiple steps. We present crystal structures of hUGDH that provide useful insight. It will be shown that protein conformational changes occurring at different time scales and level of structure have key roles in promoting the overall enzymatic reaction and are directly conducive to catalysis. Results of stopped-flow kinetic experiments enabled us to analyze oxidation of substrate alcohol and aldehyde as kinetically separated reaction steps. The data provide new evidence on the role of Cys276 as catalytic nucleophile of the reaction and lead to a comprehensive proposal for the enzymatic mechanism. Moreover, using the kinetic data, we were able to locate the rate-limiting step in the reaction of hUGDH, and this provides important information to guide the development of inhibitors for the enzyme.
      Figure thumbnail grs1
      SCHEME 1Proposed three-step enzymatic transformation of UDP-Glc into UDP-GlcUA catalyzed by UGDH.

      Acknowledgments

      We thank Dr. Kunde Guo (Structural Genomics Consortium) for contributing to protein crystallization (PDB codes 2Q3E and 2QG4) and for cloning of the hUGDH mutants. Dr. Mario Klimacek (Graz University of Technology), Dr. Panagis Filippakopoulos (Structural Genomics Consortium), Dr. Ivan Alfano (Structural Genomics Consortium), and Prof. Stefan Knapp (Structural Genomics Consortium) are gratefully acknowledged for helpful discussions and suggestions. We thank Dr. Frank von Delft (Structural Genomics Consortium) for data collection at the Swiss Light Source.

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