Crystal Structure of WbpP, a Genuine UDP-N-acetylglucosamine 4-Epimerase from Pseudomonas aeruginosa

The O antigen of lipopolysaccharide in Gram-negative bacteria plays a critical role in bacterium-host interactions, and for pathogenic bacteria it is a major virulence factor. In Pseudomonas aeruginosa serotype O6 one of the initial steps in O-antigen biosynthesis is catalyzed by a saccharide epimerase, WbpP. WbpP is a member of the UDP-hexose 4-epimerase family of enzymes and exists as a homo-dimer. This enzyme preferentially catalyzes the conversion between UDP-GlcNAc and UDPGalNAc above UDP-Glc and UDP-Gal, using NAD+ as a cofactor. The crystal structures of WbpP in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 Ǎ, respectively, which represents the first structural studies of a genuine UDP-GlcNAc 4-epimerase. These structures in combination with complementary mutagenesis studies suggest that the basis for the differential substrate specificity of WbpP is a consequence of the presence of a pliable solvent network in the active site. This information allows for a comprehensive analysis of the relationship between sequence and substrate specificity for UDP-hexose 4-epimerases and enables the formulation of consensus sequences that predict substrate specificity of UDP-hexose 4-epimerases yet to be biochemically characterized. Furthermore, the examination indicates that as little as one residue can dictate substrate specificity. Nonetheless, phylogenetic analysis suggests that this substrate specificity is an evolutionary and highly conserved property within UDP-hexose 4-epimerases.

[1]  D. Karamata,et al.  The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation. , 2003, Gene.

[2]  Dagmar Ringe,et al.  POVScript+: a program for model and data visualization using persistence of vision ray-tracing , 2003 .

[3]  Alex B. Lopez,et al.  Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis. , 2003, Molecular and biochemical parasitology.

[4]  J. R. Roper,et al.  High-resolution crystal structure of Trypanosoma brucei UDP-galactose 4'-epimerase: a potential target for structure-based development of novel trypanocides. , 2003, Molecular and biochemical parasitology.

[5]  P. Wang,et al.  New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-L-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17. , 2002, Biochemistry.

[6]  T. Roger,et al.  Initial responses to endotoxins and Gram-negative bacteria. , 2002, Clinica chimica acta; international journal of clinical chemistry.

[7]  T. Salminen,et al.  Functional Characterization of Gne (UDP-N-Acetylglucosamine- 4-Epimerase), Wzz (Chain Length Determinant), and Wzy (O-Antigen Polymerase) of Yersinia enterocolitica Serotype O:8 , 2002, Journal of bacteriology.

[8]  J. Henderson,et al.  Structural Analysis of the Y299C Mutant of Escherichia coli UDP-galactose 4-Epimerase , 2002, The Journal of Biological Chemistry.

[9]  M. Tanner,et al.  Mechanistic aspects of enzymatic carbohydrate epimerization. , 2002, Natural product reports.

[10]  J. Vanderleyden,et al.  O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. , 2001, FEMS microbiology reviews.

[11]  J. Naismith,et al.  Epimerases: structure, function and mechanism , 2001, Cellular and Molecular Life Sciences CMLS.

[12]  J. Fridovich-Keil,et al.  Human UDP-galactose 4-Epimerase , 2001, The Journal of Biological Chemistry.

[13]  W. Wakarchuk,et al.  Expression, Purification, and Biochemical Characterization of WbpP, a New UDP-GlcNAc C4 Epimerase from Pseudomonas aeruginosa Serotype O6* , 2000, The Journal of Biological Chemistry.

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

[15]  B. Beutler,et al.  Analysis of Tlr4-mediated LPS signal transduction in macrophages by mutational modification of the receptor. , 1999, Blood cells, molecules & diseases.

[16]  L. Burrows,et al.  Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide. , 1999, Microbiology.

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

[18]  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.

[19]  Jiahuai Han,et al.  Cellular activation mechanisms in septic shock. , 1998, Frontiers in bioscience : a journal and virtual library.

[20]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

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

[22]  J. Thoden,et al.  Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli. , 1997, Biochemistry.

[23]  D E Koshland,et al.  Orbital steering in the catalytic power of enzymes: small structural changes with large catalytic consequences. , 1997, Science.

[24]  P. Frey,et al.  Structural analysis of UDP-sugar binding to UDP-galactose 4-epimerase from Escherichia coli. , 1997, Biochemistry.

[25]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

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

[27]  Roderic D. M. Page,et al.  TreeView: an application to display phylogenetic trees on personal computers , 1996, Comput. Appl. Biosci..

[28]  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.

[29]  P. Frey,et al.  Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli. , 1996, Biochemistry.

[30]  J. Janin,et al.  Principles of protein-protein recognition from structure to thermodynamics. , 1995, Biochimie.

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

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

[33]  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.

[34]  Y. Knirel.  Polysaccharide antigens of Pseudomonas aeruginosa. , 1990, Critical reviews in microbiology.