Predicting protein function from structure--the roles of short-chain dehydrogenase/reductase enzymes in Bordetella O-antigen biosynthesis |
| |
Authors: | King Jerry D Harmer Nicholas J Preston Andrew Palmer Colin M Rejzek Martin Field Robert A Blundell Tom L Maskell Duncan J |
| |
Institution: | 1 Department of Veterinary Medicine, Madingley Road, University of Cambridge, Cambridge CB3 0ES, UK 2 Department of Biochemistry, 80 Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK 3 Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada N1G 2W1 4 School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK |
| |
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. |
| |
Keywords: | dTDP deoxythymidine diphosphate l-GalNAc3NAcA" target="_blank">l-GalNAc3NAcA 2 3-diacetamido-2 l-galacturonic acid" target="_blank">3-dideoxy-l-galacturonic acid GME GDP-mannose 3 5-epimerase GMER GDP-4-keto-6-deoxymannose 3 5-epimerase/reductase LPS lipopolysaccharide PDB Protein Data Bank BLAST basic local alignment search tool SDR short-chain dehydrogenase/reductase d-ManNAc3NAcA" target="_blank">UDP-d-ManNAc3NAcA UDP-2 3-diacetamido-2 d-mannuronic acid" target="_blank">3-dideoxy-d-mannuronic acid l-GalNAc3NAcA" target="_blank">UDP-l-GalNAc3NAcA UDP-2 3-diacetamido-2 l-galacturonic acid" target="_blank">3-dideoxy-l-galacturonic acid UMP uridine monophosphate MCS multiple cloning site |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|