Predicting Protein Function from Structure—The Roles of Short-chain Dehydrogenase/Reductase Enzymes in Bordetella O-antigen Biosynthesis

Abstract

The pathogenic bacteria Bordetella parapertussis and Bordetella bronchiseptica express a lipopolysaccharide O antigen containing a polymer of 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid. The O-antigen cluster contains three neighbouring genes that encode proteins belonging to the short-chain dehydrogenase/reductase (SDR) family, wbmF, wbmG and wbmH, and we aimed to elucidate their individual functions. Mutation and complementation implicate each gene in O-antigen expression but, as their putative sugar nucleotide substrates are not currently available, biochemical characterisation of WbmF, WbmG and WbmH is impractical at the present time. SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation. Because they typically share low sequence conservation, however, catalytic function cannot be predicted from sequence analysis alone. In this context, structural characterisation of the native proteins, co-crystals and small-molecule soaks enables differentiation of the functions of WbmF, WbmG and WbmH. These proteins exhibit typical SDR architecture and coordinate NAD. In the substrate-binding domain, all three enzymes bind uridyl nucleotides. WbmG contains a typical SDR catalytic TYK triad, which is required for oxidoreductase function, but the active site is devoid of additional acid–base functionality. Similarly, WbmH possesses a TYK triad, but an otherwise feature-poor active site. Consequently, 3,5-epimerase function can probably be ruled out for these enzymes. The WbmF active site contains conserved 3,5-epimerase features, namely, a positionally conserved cysteine (Cys133) and basic side chain (His90 or Asn213), but lacks the serine/threonine component of the SDR triad and therefore may not act as an oxidoreductase. The data suggest a pathway for synthesis of the O-antigen precursor UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid and illustrate the usefulness of structural data in predicting protein function.

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

“…For example, the putative C3/C5 epimerase WbmF is important for O-antigen synthesis in Bordetella species (1), and the well characterized C3/C5 epimerases RmlC from Escherichia coli and Pseudomonas aeruginosa and RfbC from Salmonella enterica are involved in the production of dTDP-L-rhamnose for O-antigen synthesis (2)(3)(4)(5). Likewise, GDP-4-keto-6-deoxy-␣-D-mannose C3/C5 epimerases/C4 reductases such as GFS and GMER involved in making GDP-L-fucose from GDP-mannose have been studied extensively as the process of synthesis of GDP-L-fucose is well conserved throughout evolution (6 -8) and L-fucose is important in a variety of cellular processes.…”

Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

“…Both organisms also produce D-ManNAc3NAcA, and 2,3-diacetamido-2,3-dideoxy-L-galactosamine (L-GalNAc3NAcA), as constituents of the LPS (23,24). Because UDP-LGalNAc3NAcA is thought to be synthesized from UDP-DManNAc3NAcA by the enzymes of the wbm* gene cluster (25), further studies will benefit from enzymatic production of the predicted substrate. Purified UDP-D-GlcNAc3NA will also be made available to the community to support enzyme specificity tests or other applications, as reported for UDP-DGlcNAc3NAcA (26).…”

Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

“…The O antigen linker also contains ManNAc3NAcAN, a uronamide derivative of ManNAc3NAcA, and GlcNAc3NAcAN, a uronamide derivative of an intermediate from the ManNAc3NAcA pathway (34). The Bordetella O antigen produced by B. bronchiseptica and B. parapertussis contains L-GalNAc3NAcA and L-GalNAc3NAcAN, believed to be derivatives of UDP-D-ManNAc3NAcA synthesized by enzymes of the wbm locus (21,34). Based on our hypothesis, an active copy of either of the wbpO genes should be essential for the production of all these uronic acid sugars.…”

“…B. bronchiseptica and B. parapertussis LPS also contains a repeating polysaccharide known as the O antigen (12). The O antigen contains 2,3-diacetamido-2,3-dideoxy-L-galactosamine (L-GalNAc3NAcA) (33), which is thought to be synthesized from UDP-D-ManNAc3NAcA by the enzymes of the wbm gene cluster (21). In B. pertussis, the LPS is not capped with the O antigen due to the deletion of the wbm cluster (33).…”