Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid,

摘要

2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugarnfirst identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has sincenbeen observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough.nFive enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-nD-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzesnthe second step in the pathway, namely, the oxidation of the C-30nhydroxyl group on the UDP-linked sugar tona keto moiety and the reduction of NADþ to NADH. This enzyme has been shown to use R-ketoglutarate asnan oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures werendetermined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and R-ketoglutarate,nand the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). Thentetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely tonone another. Both R-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbonnatoms (C-2 and C-30n, respectively) abutting the re face of the cofactor. They are positioned ∼3A from thennicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation whennbound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.