title: Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis.
abstract: CDP-D-glucose 4,6-dehydratase catalyzes the conversion of CDP-D-glucose to CDP-4-keto-6-deoxyglucose in an NAD(+)-dependent manner. The product of this conversion is a building block for a variety of primary antigenic determinants in bacteria, possibly implicated directly in reactive arthritis. Here, we describe the solution of the high-resolution crystal structure of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis in the resting state. This structure represents the first CDP nucleotide utilizing dehydratase of the short-chain dehydrogenase/reductase (SDR) family to be determined, as well as the first tetrameric structure of the subfamily of SDR enzymes in which NAD(+) undergoes a full reaction cycle. On the basis of a comparison of this structure with structures of homologous enzymes, a chemical mechanism is proposed in which Tyr157 acts as the catalytic base, initiating hydride transfer by abstraction of the proton from the sugar 4'-hydroxyl. Concomitant with the removal of the proton from the 4'-hydroxyl oxygen, the sugar 4'-hydride is transferred to the B face of the NAD(+) cofactor, forming the reduced cofactor and a CDP-4-keto-d-glucose intermediate. A conserved Lys161 most likely acts to position the NAD(+) cofactor so that hydride transfer is favorable and/or to reduce the pK(a) of Tyr157. Following substrate oxidation, we propose that Lys134, acting as a base, would abstract the 5'-hydrogen of CDP-4-keto-D-glucose, priming the intermediate for the spontaneous loss of water. Finally, the resulting Delta(5,6)-glucoseen intermediate would be reduced suprafacially by the cofactor, and reprotonation at C-5' is likely mediated by Lys134.
authors.affiliation: Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454-9110, USA. vogan@crystal.harvard.edu
authors.forename: Erik M
authors.initials: EM
authors.lastname: Vogan
authors.forename: Cornelia
authors.initials: C
authors.lastname: Bellamacina
authors.forename: Xuemei
authors.initials: X
authors.lastname: He
authors.forename: Hung-wen
authors.initials: HW
authors.lastname: Liu
authors.forename: Dagmar
authors.initials: D
authors.lastname: Ringe
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Petsko
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014534:UDPglucose 4-Epimerase / Q000737:chemistry; D015011:Yersinia pseudotuberculosis / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D006836:Hydro-Lyases; C089119:CDP-glucose 4,6-dehydratase; D014534:UDPglucose 4-Epimerase