title: Structure and mechanism of human UDP-glucose 6-dehydrogenase.
abstract: 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, Tyr(14), Glu(161), and Glu(165) 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 Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) 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.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Egger
authors.forename: Apirat
authors.initials: A
authors.lastname: Chaikuad
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Kavanagh
authors.forename: Udo
authors.initials: U
authors.lastname: Oppermann
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003067:Coenzymes; D009243:NAD; D014533:Uridine Diphosphate Glucose Dehydrogenase