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Predicting protein function from structure--the roles of short-chain dehydrogenase/reductase enzymes in Bordetella O-antigen biosynthesis.

King JD, Harmer NJ, Preston A, Palmer CM, Rejzek M, Field RA, Blundell TL, Maskell DJ - J. Mol. Biol. (2007)

Bottom Line: SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Medicine, Madingley Road, University of Cambridge, Cambridge CB3 0ES, UK. jking01@uoguelph.ca

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.

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Proposed pathway for the synthesis of UDP-l-GalNAc3NAcA catalysed by WbmF, WbmG and WbmH.
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fig8: Proposed pathway for the synthesis of UDP-l-GalNAc3NAcA catalysed by WbmF, WbmG and WbmH.

Mentions: We therefore propose the following biosynthetic pathway (Fig. 8) for the 3,5-epimerisation of UDP-d-ManNAc3NAcA: One of the putative oxidoreductases, WbmG or WbmH, catalyses the initial oxidation at C-4, and the other oxidoreductase is responsible for the final reduction step. Introduction of the keto group lowers the pKa of the C-3 and C-5 hydrogen atoms, allowing WbmF to catalyse proton exchange effecting 3,5-epimerisation. This scheme is the most consistent with all of the available data. The function suggested for WbmF is very unusual in that we suggest that this enzyme does not catalyse oxidation or reduction of its substrate. If this is the case, WbmF would be the first such member of the SDR family, and this would imply that the cofactor in WbmF has a purely structural role, without participating in the catalytic cycle. It has been suggested that the transcriptional regulation protein NmrA from Aspergillus nidulans is a NAD-binding SDR family member that is not an enzyme at all. NmrA appears to function in control of nitrogen metabolism through physical interaction with the GATA family transcription factor AreA, possibly by controlling the rate of entry of AreA into the nucleus,33 and the structural equivalents of the SDR triad tyrosine and serine/threonine in NmrA are Met127 and Met113, respectively.34 Since the NmrA structure was reported, however, several SDR enzymes with SMK triads have been reported (e.g., WbpM from P. aeruginosa),26 so that the nonconservation of the SDR catalytic triad in NmrA should not rule out an enzyme function as absolutely as first appeared.34 An alternative possibility to the biosynthetic pathway we have outlined (Fig. 8) is that WbmF does in fact catalyse either oxidation or reduction as well as the 3,5-epimerisation, in which case the third SDR may participate in O-antigen expression by performing a function in the synthesis of the complicated sugar residue that adorns the nonreducing terminus of the O-chain.35 The nonredox role we propose for WbmF is consistent, however, with the lack of the conserved SDR triad in this enzyme, and if this protein operates purely as a 3,5-epimerase this may also suggest a mechanism by which the wbmF mutant is able to express O antigen, albeit in greatly reduced amounts. The product of the initial oxidation reaction is activated for proton exchange α- to the keto group and it may be that racemisation at these positions occurs at a biologically significant rate without an absolute requirement for enzyme catalysis.


Predicting protein function from structure--the roles of short-chain dehydrogenase/reductase enzymes in Bordetella O-antigen biosynthesis.

King JD, Harmer NJ, Preston A, Palmer CM, Rejzek M, Field RA, Blundell TL, Maskell DJ - J. Mol. Biol. (2007)

Proposed pathway for the synthesis of UDP-l-GalNAc3NAcA catalysed by WbmF, WbmG and WbmH.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2279256&req=5

fig8: Proposed pathway for the synthesis of UDP-l-GalNAc3NAcA catalysed by WbmF, WbmG and WbmH.
Mentions: We therefore propose the following biosynthetic pathway (Fig. 8) for the 3,5-epimerisation of UDP-d-ManNAc3NAcA: One of the putative oxidoreductases, WbmG or WbmH, catalyses the initial oxidation at C-4, and the other oxidoreductase is responsible for the final reduction step. Introduction of the keto group lowers the pKa of the C-3 and C-5 hydrogen atoms, allowing WbmF to catalyse proton exchange effecting 3,5-epimerisation. This scheme is the most consistent with all of the available data. The function suggested for WbmF is very unusual in that we suggest that this enzyme does not catalyse oxidation or reduction of its substrate. If this is the case, WbmF would be the first such member of the SDR family, and this would imply that the cofactor in WbmF has a purely structural role, without participating in the catalytic cycle. It has been suggested that the transcriptional regulation protein NmrA from Aspergillus nidulans is a NAD-binding SDR family member that is not an enzyme at all. NmrA appears to function in control of nitrogen metabolism through physical interaction with the GATA family transcription factor AreA, possibly by controlling the rate of entry of AreA into the nucleus,33 and the structural equivalents of the SDR triad tyrosine and serine/threonine in NmrA are Met127 and Met113, respectively.34 Since the NmrA structure was reported, however, several SDR enzymes with SMK triads have been reported (e.g., WbpM from P. aeruginosa),26 so that the nonconservation of the SDR catalytic triad in NmrA should not rule out an enzyme function as absolutely as first appeared.34 An alternative possibility to the biosynthetic pathway we have outlined (Fig. 8) is that WbmF does in fact catalyse either oxidation or reduction as well as the 3,5-epimerisation, in which case the third SDR may participate in O-antigen expression by performing a function in the synthesis of the complicated sugar residue that adorns the nonreducing terminus of the O-chain.35 The nonredox role we propose for WbmF is consistent, however, with the lack of the conserved SDR triad in this enzyme, and if this protein operates purely as a 3,5-epimerase this may also suggest a mechanism by which the wbmF mutant is able to express O antigen, albeit in greatly reduced amounts. The product of the initial oxidation reaction is activated for proton exchange α- to the keto group and it may be that racemisation at these positions occurs at a biologically significant rate without an absolute requirement for enzyme catalysis.

Bottom Line: SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Medicine, Madingley Road, University of Cambridge, Cambridge CB3 0ES, UK. jking01@uoguelph.ca

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.

Show MeSH