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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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A model of the proposed substrate in the binding site of WbmF. (a) Overview of modelled interaction of WbmF with the 4-keto derivative of UDP-d-ManNAc3NAcA. Protein is shown as cartoon, with key (labelled) side chains, NAD and sugar nucleotide in sticks. The elements of the UDP-sugar found in the experimental structure have paler colours to highlight the modelled sections. (b) and (c) Close-up of the modelled sugar ring (shown as spheres) demonstrates that there are no clashes between the proposed ligand and the protein surface. The two views show opposite sides of the sugar: (c) shows the surface with the imaged slab cut at the level of the first sugar atom, with NAD surface also removed for clarity. Carbon atoms are yellow for protein, white for the NAD and cyan for 4-keto UDP-d-ManNAc3NAcA; oxygen is red, nitrogen is blue, phosphorus is orange and sulfur is purple.
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fig7: A model of the proposed substrate in the binding site of WbmF. (a) Overview of modelled interaction of WbmF with the 4-keto derivative of UDP-d-ManNAc3NAcA. Protein is shown as cartoon, with key (labelled) side chains, NAD and sugar nucleotide in sticks. The elements of the UDP-sugar found in the experimental structure have paler colours to highlight the modelled sections. (b) and (c) Close-up of the modelled sugar ring (shown as spheres) demonstrates that there are no clashes between the proposed ligand and the protein surface. The two views show opposite sides of the sugar: (c) shows the surface with the imaged slab cut at the level of the first sugar atom, with NAD surface also removed for clarity. Carbon atoms are yellow for protein, white for the NAD and cyan for 4-keto UDP-d-ManNAc3NAcA; oxygen is red, nitrogen is blue, phosphorus is orange and sulfur is purple.

Mentions: The analysis of the potential catalytic chemistry of WbmF suggested that its role in O-antigen biosynthesis may be to catalyse the 3,5-epimerisation required in the overall conversion of UDP-d-ManNAc3NAcA to UDP-l-GalNAc3NAcA. The 4-keto derivative of UDP-d-ManNAc3NAcA is therefore a likely substrate or reaction intermediate for WbmF. This compound contains a bulkier sugar than any of the substrates of the characterised sugar-nucleotide-modifying SDRs. An important test of our proposed pathway is that the enzymes must be able to accommodate these unusually large substrates. We modelled 4-keto UDP-d-ManNAc3NAcA into the active site of WbmF (Fig. 7). This showed that it is feasible for the substrate to occupy the active site of WbmF in a manner that is consistent with the experimentally determined binding site for UMP, the proximity of the most likely catalytic site, and good geometry.


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)

A model of the proposed substrate in the binding site of WbmF. (a) Overview of modelled interaction of WbmF with the 4-keto derivative of UDP-d-ManNAc3NAcA. Protein is shown as cartoon, with key (labelled) side chains, NAD and sugar nucleotide in sticks. The elements of the UDP-sugar found in the experimental structure have paler colours to highlight the modelled sections. (b) and (c) Close-up of the modelled sugar ring (shown as spheres) demonstrates that there are no clashes between the proposed ligand and the protein surface. The two views show opposite sides of the sugar: (c) shows the surface with the imaged slab cut at the level of the first sugar atom, with NAD surface also removed for clarity. Carbon atoms are yellow for protein, white for the NAD and cyan for 4-keto UDP-d-ManNAc3NAcA; oxygen is red, nitrogen is blue, phosphorus is orange and sulfur is purple.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: A model of the proposed substrate in the binding site of WbmF. (a) Overview of modelled interaction of WbmF with the 4-keto derivative of UDP-d-ManNAc3NAcA. Protein is shown as cartoon, with key (labelled) side chains, NAD and sugar nucleotide in sticks. The elements of the UDP-sugar found in the experimental structure have paler colours to highlight the modelled sections. (b) and (c) Close-up of the modelled sugar ring (shown as spheres) demonstrates that there are no clashes between the proposed ligand and the protein surface. The two views show opposite sides of the sugar: (c) shows the surface with the imaged slab cut at the level of the first sugar atom, with NAD surface also removed for clarity. Carbon atoms are yellow for protein, white for the NAD and cyan for 4-keto UDP-d-ManNAc3NAcA; oxygen is red, nitrogen is blue, phosphorus is orange and sulfur is purple.
Mentions: The analysis of the potential catalytic chemistry of WbmF suggested that its role in O-antigen biosynthesis may be to catalyse the 3,5-epimerisation required in the overall conversion of UDP-d-ManNAc3NAcA to UDP-l-GalNAc3NAcA. The 4-keto derivative of UDP-d-ManNAc3NAcA is therefore a likely substrate or reaction intermediate for WbmF. This compound contains a bulkier sugar than any of the substrates of the characterised sugar-nucleotide-modifying SDRs. An important test of our proposed pathway is that the enzymes must be able to accommodate these unusually large substrates. We modelled 4-keto UDP-d-ManNAc3NAcA into the active site of WbmF (Fig. 7). This showed that it is feasible for the substrate to occupy the active site of WbmF in a manner that is consistent with the experimentally determined binding site for UMP, the proximity of the most likely catalytic site, and good geometry.

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