|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 25, 19060-19067, June 23, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Microbiology, University of Guelph,
Guelph, Ontario N1G 2W1 and the ¶ Institute for Biological
Sciences, National Research Council,
Ottawa, Ontario K1A 0R6, Canada
B-band lipopolysaccharide is an important
virulence factor of the opportunistic pathogen Pseudomonas
aeruginosa. WbpP is an enzyme essential for B-band
lipopolysaccharide production in serotype O6. Sequence analysis
suggests that it is involved in the formation of
N-acetylgalacturonic acid. To test this hypothesis,
overexpression and biochemical characterization of WbpP were performed.
By using spectrophotometric assays and capillary electrophoresis, we
show that WbpP is a UDP-GlcNAc C4 epimerase. The Km
for UDP-GlcNAc and UDP-GalNAc are 197 and 224 µM,
respectively. At equilibrium, 70% of UDP-GalNAc is converted to
UDP-GlcNAc, whereas the yield of the reverse reaction is only 30%. The
enzyme can also catalyze the inter-conversion of non-acetylated
substrates, although the efficiency of catalysis is significantly
lower. Only 15 and 40% of UDP-Glc and UDP-Gal, respectively, are
converted at equilibrium. WbpP contains tightly bound NAD(H) and does
not require additional cofactors for activity. It exists as a dimer in
its native state. This paper is the first report of expression and
characterization of a C4 UDP-GlcNAc epimerase at the biochemical level.
Moreover, the characterization of the enzymatic function of WbpP will
help clarify ambiguous surface carbohydrate biosynthetic pathways in P. aeruginosa and other organisms where homologues of WbpP exist.
Expression, Purification, and Biochemical Characterization of
WbpP, a New UDP-GlcNAc C4 Epimerase from Pseudomonas
aeruginosa Serotype O6*
,§,
*
This work was supported in part by Grant MT14687 from the
Medical Research Council of Canada (to J. S. L.) and the Canadian Bacterial Diseases Network (a consortium of the Federal Networks of
Centres of Excellence).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of postdoctoral fellowships from the Canadian Cystic
Fibrosis Foundation.
§
Current address: College of Veterinary Medicine, Dept. of
Pathobiology, University of Florida, P. O. Box 110880, Gainesville, FL
32611-0880.
To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 3823); Fax: 519-837-1802; E-mail: jlam@uoguelph.ca.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. L. Westman, A. Preston, R. A. Field, and J. S. Lam Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters J. Bacteriol., September 15, 2008; 190(18): 6060 - 6069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Valiente, N. Jimenez, S. Merino, J. M. Tomas, and C. Amaro Vibrio vulnificus Biotype 2 Serovar E gne but Not galE Is Essential for Lipopolysaccharide Biosynthesis and Virulence Infect. Immun., April 1, 2008; 76(4): 1628 - 1638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. Miller, M. J. Matewish, D. J. McNally, N. Ishiyama, E. M. Anderson, D. Brewer, J.-R. Brisson, A. M. Berghuis, and J. S. Lam Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO J. Biol. Chem., February 8, 2008; 283(6): 3507 - 3518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Cunneen and P. R. Reeves The Yersinia kristensenii O11 O-Antigen Gene Cluster was Acquired by Lateral Gene Transfer and Incorporated at a Novel Chromosomal Locus Mol. Biol. Evol., June 1, 2007; 24(6): 1355 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Canals, N. Jimenez, S. Vilches, M. Regue, S. Merino, and J. M. Tomas Role of Gne and GalE in the Virulence of Aeromonas hydrophila Serotype O34 J. Bacteriol., January 15, 2007; 189(2): 540 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Vijayakumar, A. Merkx-Jacques, D. B. Ratnayake, I. Gryski, R. K. Obhi, S. Houle, C. M. Dozois, and C. Creuzenet Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni J. Biol. Chem., September 22, 2006; 281(38): 27733 - 27743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Canals, N. Jimenez, S. Vilches, M. Regue, S. Merino, and J. M. Tomas The UDP N-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic Aeromonas hydrophila Serotype O34 Virulence Infect. Immun., January 1, 2006; 74(1): 537 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Y. Park, J. H. Lee, M. W. Kim, H. G. Jeong, B. C. Lee, T. S. Kim, and S. H. Choi Identification of the Vibrio vulnificus wbpP Gene and Evaluation of Its Role in Virulence Infect. Immun., January 1, 2006; 74(1): 721 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Frirdich and C. Whitfield Characterization of GlaKP, a UDP-Galacturonic Acid C4-Epimerase from Klebsiella pneumoniae with Extended Substrate Specificity J. Bacteriol., June 15, 2005; 187(12): 4104 - 4115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Obhi and C. Creuzenet Biochemical Characterization of the Campylobacter jejuni Cj1294, a Novel UDP-4-keto-6-deoxy-GlcNAc Aminotransferase That Generates UDP-4-amino-4,6-dideoxy-GalNAc J. Biol. Chem., May 27, 2005; 280(21): 20902 - 20908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bernatchez, C. M. Szymanski, N. Ishiyama, J. Li, H. C. Jarrell, P. C. Lau, A. M. Berghuis, N. M. Young, and W. W. Wakarchuk A Single Bifunctional UDP-GlcNAc/Glc 4-Epimerase Supports the Synthesis of Three Cell Surface Glycoconjugates in Campylobacter jejuni J. Biol. Chem., February 11, 2005; 280(6): 4792 - 4802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. L. Miller, C. Q. Wenzel, C. Daniels, S. Larocque, J.-R. Brisson, and J. S. Lam Biochemical Characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1 J. Biol. Chem., September 3, 2004; 279(36): 37551 - 37558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Sweet, A. A. Ribeiro, and C. R. H. Raetz Oxidation and Transamination of the 3''-Position of UDP-N-Acetylglucosamine by Enzymes from Acidithiobacillus ferrooxidans: ROLE IN THE FORMATION OF LIPID A MOLECULES WITH FOUR AMIDE-LINKED ACYL CHAINS J. Biol. Chem., June 11, 2004; 279(24): 25400 - 25410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Ishiyama, C. Creuzenet, J. S. Lam, and A. M. Berghuis Crystal Structure of WbpP, a Genuine UDP-N-acetylglucosamine 4-Epimerase from Pseudomonas aeruginosa: SUBSTRATE SPECIFICITY IN UDP-HEXOSE 4-EPIMERASES J. Biol. Chem., May 21, 2004; 279(21): 22635 - 22642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Merkx-Jacques, R. K. Obhi, G. Bethune, and C. Creuzenet The Helicobacter pylori flaA1 and wbpB Genes Control Lipopolysaccharide and Flagellum Synthesis and Function J. Bacteriol., April 15, 2004; 186(8): 2253 - 2265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-Q. Xu, J. Thompson, and J. O. Cisar Genetic Loci for Coaggregation Receptor Polysaccharide Biosynthesis in Streptococcus gordonii 38 J. Bacteriol., September 15, 2003; 185(18): 5419 - 5430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Creuzenet, R. V. Urbanic, and J. S. Lam Structure-Function Studies of Two Novel UDP-GlcNAc C6 Dehydratases/C4 Reductases. VARIATION FROM THE SYK DOGMA J. Biol. Chem., July 19, 2002; 277(30): 26769 - 26778. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. Raymond, E. H. Sims, A. Kas, D. H. Spencer, T. V. Kutyavin, R. G. Ivey, Y. Zhou, R. Kaul, J. B. Clendenning, and M. V. Olson Genetic Variation at the O-Antigen Biosynthetic Locus in Pseudomonas aeruginosa J. Bacteriol., July 1, 2002; 184(13): 3614 - 3622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wang, S. Huskic, A. Cisterne, D. Rothemund, and P. R. Reeves The O-Antigen Gene Cluster of Escherichia coli O55:H7 and Identification of a New UDP-GlcNAc C4 Epimerase Gene J. Bacteriol., May 15, 2002; 184(10): 2620 - 2625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. McKerral and R. Y. C. Lo Construction and Characterization of an Acapsular Mutant of Mannheimia haemolytica A1 Infect. Immun., May 1, 2002; 70(5): 2622 - 2629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhao, C. Creuzenet, M. Belanger, E. Egbosimba, J. Li, and J. S. Lam WbpO, a UDP-N-acetyl-D-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6 J. Biol. Chem., October 20, 2000; 275(43): 33252 - 33259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Creuzenet, M. J. Schur, J. Li, W. W. Wakarchuk, and J. S. Lam FlaA1, a New Bifunctional UDP-GlcNAc C6 Dehydratase/ C4 Reductase from Helicobacter pylori J. Biol. Chem., November 3, 2000; 275(45): 34873 - 34880. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |