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[摘要]：2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylD-glucosamine. Here we describe a structural study of W1bA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3' hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD+ to NADH. This enzyme has been shown to use alpha-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and la-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both a-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3', respectively) abutting the re face of the cofactor. They are positioned similar to 3 angstrom from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the W1bA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis. |
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