Expression, purification, and biochemical characterization of WbpP, a new UDP-GlcNAc C4 epimerase from Pseudomonas aeruginosa serotype O6.
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 K(m) for UDP-GlcNAc and UDP-GalNAc are 197 and 224 micrometer, 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.
Creuzenet
C
C
Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Belanger
M
M
Wakarchuk
W W
WW
Lam
J S
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity
D002413:Cations, Divalent; D017931:DNA Primers; D009243:NAD; D002238:Carbohydrate Epimerases; C023223:UDP-N-acetylglucosamine 4-epimerase