Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis.

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.

[1]  K D Cowtan,et al.  Improvement of macromolecular electron-density maps by the simultaneous application of real and reciprocal space constraints. , 1993, Acta crystallographica. Section D, Biological crystallography.

[2]  M. Karplus,et al.  Crystallographic refinement by simulated annealing: application to crambin , 1989 .

[3]  P. Frey,et al.  UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition. , 1996, Biochemistry.

[4]  L. Lally.  The CCP 4 Suite — Computer programs for protein crystallography , 1998 .

[5]  M. Baker,et al.  Expansion of the mammalian 3β‐hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP‐galactose‐4‐epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus , 1992, FEBS letters.

[6]  A. Brünger.  Crystallographic refinement by simulated annealing. Application to a 2.8 A resolution structure of aspartate aminotransferase. , 1988, Journal of molecular biology.

[7]  J. Thorson,et al.  Probing the coenzyme and substrate binding events of CDP-D-glucose 4,6-dehydratase: mechanistic implications. , 1996, Biochemistry.

[8]  P. Frey,et al.  DTDP-GLUCOSE 4,6-DEHYDRATASE FROM E. COLI , 1998 .

[9]  M Krook,et al.  Short-chain dehydrogenases/reductases (SDR). , 1995, Biochemistry.

[10]  K Cowtan,et al.  Miscellaneous algorithms for density modification. , 1998, Acta crystallographica. Section D, Biological crystallography.

[11]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[12]  A. Brunger.  Crystallographic refinement by simulated annealing , 1988 .

[13]  C. Walsh,et al.  Enzymatic Reaction Mechanisms , 1978 .

[14]  J. Strominger,et al.  Enzymatic synthesis of cytidine diphosphate 3,6-dideoxyhexoses. 3. Cytidine diphosphate D-glucose oxidoreductase. , 1966, The Journal of biological chemistry.

[15]  Xiangshu Jin,et al.  Structures of NAD(+)- and NADH-bound 1-l-myo-inositol 1-phosphate synthase. , 2003, Acta crystallographica. Section D, Biological crystallography.

[16]  P. Frey,et al.  Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli. , 1997, Biochemistry.

[17]  Chris Sander,et al.  The double cubic lattice method: Efficient approaches to numerical integration of surface area and volume and to dot surface contouring of molecular assemblies , 1995, J. Comput. Chem..

[18]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[19]  B. C. Wang.  Resolution of phase ambiguity in macromolecular crystallography. , 1985, Methods in enzymology.

[20]  R. D. Bevill.  The mechanism of action of CDP-glucose oxidoreductase. , 1968, Biochemical and biophysical research communications.

[21]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[22]  J. Fridovich-Keil,et al.  Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase. , 2000, Biochemistry.

[23]  P. Reeves,et al.  Relationships among the rfb regions of Salmonella serovars A, B, and D , 1991, Journal of bacteriology.

[24]  Hung‐wen Liu,et al.  Stereospecificity of hydride transfer for the catalytically recycled NAD+ in CDP-d-glucose 4,6-dehydratase , 1998 .

[25]  Frank Eisenhaber,et al.  Improved strategy in analytic surface calculation for molecular systems: Handling of singularities and computational efficiency , 1993, J. Comput. Chem..

[26]  A. Elbein,et al.  THE BIOSYNTHESIS OF CELL WALL LIPOPOLYSACCHARIDE IN ESCHERICHIA COLI. II. GUANOSINE DIPHOSPHATE 4-KETO-6-DEOXY-D-MANNOSE, AN INTERMEDIATE IN THE BIOSYNTHESIS OF GUANOSINE DIPHOSPHATE COLITOSE. , 1965, The Journal of biological chemistry.

[27]  J. Strominger,et al.  Enzymatic synthesis of cytidine diphosphate 3,6-dideoxyhexoses. I. Over-all reactions. , 1966, Journal of Biological Chemistry.

[28]  G. Petsko,et al.  Tyrosine-48 is the proton donor and histidine-110 directs substrate stereochemical selectivity in the reduction reaction of human aldose reductase: enzyme kinetics and crystal structure of the Y48H mutant enzyme. , 1994, Biochemistry.

[29]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[30]  E. Lattman,et al.  Representation of phase probability distributions for simplified combination of independent phase information , 1970 .

[31]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[32]  J. Strominger,et al.  Enzymatic Synthesis of Cytidine Diphosphate 3,6-Dideoxyhexoses IV. PARTIAL RESOLUTION OF THE ENZYMES REQUIRED FOR REDUCTION OF CYTIDINE DIPHOSPHATE 4-KETO-6-DEOXY-d-GLUCOSE AND ISOLATION OF A NEW INTERMEDIATE , 1967 .

[33]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[34]  J. Thoden,et al.  Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli. , 1998, Biochemistry.

[35]  J. Thorson,et al.  Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA , 1994, Journal of bacteriology.

[36]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[37]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[38]  P. Frey,et al.  Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism. , 1996, Biochemistry.

[39]  C. Bellamacina.  The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[40]  J. R. Somoza,et al.  Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose. , 2000, Structure.

[41]  R. Lahesmaa,et al.  Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with or without reactive arthritis. , 1998, Arthritis and rheumatism.

[42]  Anders Liljas,et al.  2 Evolutionary and Structural Relationships among Dehydrogenases , 1975 .

[43]  Chris Sander,et al.  Three sisters, different names , 1994, Nature Structural Biology.

[44]  P. Howell,et al.  Identification of heavy‐atom derivatives by normal probability methods , 1992 .

[45]  Hung‐wen Liu,et al.  Formation of unusual sugars: mechanistic studies and biosynthetic applications. , 2002, Annual review of biochemistry.

[46]  P. Reeves,et al.  Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6-dideoxyhexose abequose. , 1991, Journal of general microbiology.

[47]  L. Glaser,et al.  16 Dehydration in Nucleotide-Linked Deoxysugar Synthesis , 1971 .

[48]  P. Frey,et al.  Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site. , 2001, Biochemistry.

[49]  R. Jenness,et al.  Stereospecificity of L-myo-inositol-1-phosphate synthase for nicotinamide adenine dinucleotide. , 1981, Biochemistry.

[50]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[51]  P. Reeves,et al.  O-antigen variation in Salmonella spp.: rfb gene clusters of three strains , 1988, Journal of bacteriology.

[52]  P. Frey,et al.  Identification of lysine 153 as a functionally important residue in UDP-galactose 4-epimerase from Escherichia coli. , 1993, Biochemistry.

[53]  S. Kirkwood,et al.  The mechanism of action of the enzyme uridine diphosphoglucose 4-epimerase. Proof of an oxidation-reduction mechanism with direct transfer of hydrogen between substrate and the B-position of the enzyme-bound pyridine nucleotide. , 1971, The Journal of biological chemistry.

[54]  G. Petsko,et al.  Purification, crystallization and molecular symmetry of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis. , 2002, Acta crystallographica. Section D, Biological crystallography.

[55]  J. Strominger,et al.  Enzymatic synthesis of cytidine diphosphate ascarylose. , 1964, Biochemical and biophysical research communications.

[56]  P A Frey,et al.  The molecular structure of UDP‐galactose 4‐epimerase from Escherichia coli determined at 2.5 Å resolution , 1992, Proteins.

[57]  Christopher P. Bonin,et al.  Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity. , 2002, Biochemistry.

[58]  J. Kraut,et al.  CHYMOTRYPSINOGEN: A THREE-DIMENSIONAL FOURIER SYNTHESIS AT 5 å RESOLUTION , 1962 .

[59]  Erik Nordling,et al.  Short-chain dehydrogenases/reductases (SDR): the 2002 update. , 2003, Chemico-biological interactions.

[60]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[61]  C. Whitfield,et al.  The crystal structure of dTDP-D-Glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar Typhimurium, the second enzyme in the dTDP-l-rhamnose pathway. , 2001, Journal of molecular biology.

[62]  R. N. Russell,et al.  Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase. , 1992, The Journal of biological chemistry.

[63]  A. Brünger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures , 1992, Nature.