Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

摘要

The unusual sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, or ManNAc3NAcA, hasnbeen observed in the lipopolysaccharides of both pathogenic and nonpathogenicGram-negative bacteria. It isnadded to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates UDP-nManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcAnstarting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene andnreferred to asWlbA, catalyzes the NAD-dependent oxidation of the C-30nhydroxyl group of the UDP-linkednsugar. Here we describe a combined structural and functional investigation of the WlbA enzymes fromnBordetella pertussis and Chromobacterium violaceum. For this investigation, ternary structures werendetermined in the presence of NAD(H) and substrate to 2.13 and 1.5 A resolution, respectively. Both ofnthe enzymes display octameric quaternary structures with their active sites positioned far apart. The octamersncan be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for thesenenzymes are sequential and that they do not require R-ketoglutarate for activity. These results are in sharpncontrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermusnthermophilus, which function via ping-pong mechanisms that involve R-ketoglutarate. Taken together, thenresults reported here demonstrate that there are two distinct families of WlbA enzymes, which differ withnrespect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms.