Identification and characterization of NeuB3 from Campylobacter jejuni as a pseudaminic acid synthase.
Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea worldwide, and Helicobacter pylori is known to cause duodenal ulcers. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with a 2-keto-3-deoxy acid, pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid). The presence of pseudaminic acid is required for the proper development of the flagella and is thereby necessary for motility in, and invasion of, the host. In this study we report the first characterization of NeuB3 from C. jejuni as a pseudaminic acid synthase; the enzyme directly responsible for the biosynthesis of pseudaminic acid. Pseudaminic acid synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with the hexose, 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc), to form pseudaminic acid and phosphate. The enzymatic activity was monitored using 1H and 31P NMR spectroscopy, and the product was isolated and characterized. Kinetic analysis reveals that pseudaminic acid synthase requires the presence of a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. A coupled enzymatic assay gave the values for k(cat) of 0.65 +/- 0.01 s(-1), K(m)PEP of 6.5 +/- 0.4 microM, and K(m)6-deoxy-AltdiNAc of 9.5 +/- 0.7 microM. A mechanistic study on pseudaminic acid synthase, using [2-18O]PEP, shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing synthases such as sialic acid synthase.
Chou
Wayne K
WK
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Dick
Scott
S
Wakarchuk
Warren W
WW
Tanner
Martin E
ME
D017000:Campylobacter coli / Q000378:metabolism; D016123:Campylobacter jejuni / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry* / Q000502:physiology; D007652:Oxo-Acid-Lyases / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010728:Phosphoenolpyruvate / Q000737:chemistry
D010710:Phosphates; D003035:Cobalt; D010728:Phosphoenolpyruvate; C018730:N-acetylneuraminate synthase; D000216:N-Acylneuraminate Cytidylyltransferase; D007652:Oxo-Acid-Lyases