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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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Comparison of active-site residues in WbmF, WbmG and WbmH compared with WbpP25 and GMER. (a) The SDR catalytic triad is conserved in WbmG and WbmH but not in WbmF where the residue normally found as serine or threonine superimposes onto the position of Ala131. (b) GMER is shown as an example of an SDR that catalyses 3,5-epimerisation of its substrate.21 The GMER epimerase catalytic residues Cys109 and His179 superimpose onto hydrophobic residues in WbmG and WbmH. WbmF has side chains in these positions (Cys133 and Asn213) that may be capable of acid–base chemistry. *Cys133 is not resolved in the WbmF crystal; its position here is inferred from the neighbouring residue (Gly132), which is visible in the electron density.
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fig6: Comparison of active-site residues in WbmF, WbmG and WbmH compared with WbpP25 and GMER. (a) The SDR catalytic triad is conserved in WbmG and WbmH but not in WbmF where the residue normally found as serine or threonine superimposes onto the position of Ala131. (b) GMER is shown as an example of an SDR that catalyses 3,5-epimerisation of its substrate.21 The GMER epimerase catalytic residues Cys109 and His179 superimpose onto hydrophobic residues in WbmG and WbmH. WbmF has side chains in these positions (Cys133 and Asn213) that may be capable of acid–base chemistry. *Cys133 is not resolved in the WbmF crystal; its position here is inferred from the neighbouring residue (Gly132), which is visible in the electron density.

Mentions: SDR enzymes have a conserved catalytic triad that is involved in their oxidoreductase activity and that is classically composed of a spatially conserved serine, tyrosine and lysine (SYK) triad, for example, Ser142, Tyr166 and Lys170 in WbpP. The first member of this triad is sometimes found as a threonine (TYK),23 and the tyrosine can be replaced by a methionine (SMK).26 The SDR triad is conserved in WbmG and WbmH as TYK but in WbmF Ala131 superimposes onto the Ser/Thr position (Fig. 6a).


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)

Comparison of active-site residues in WbmF, WbmG and WbmH compared with WbpP25 and GMER. (a) The SDR catalytic triad is conserved in WbmG and WbmH but not in WbmF where the residue normally found as serine or threonine superimposes onto the position of Ala131. (b) GMER is shown as an example of an SDR that catalyses 3,5-epimerisation of its substrate.21 The GMER epimerase catalytic residues Cys109 and His179 superimpose onto hydrophobic residues in WbmG and WbmH. WbmF has side chains in these positions (Cys133 and Asn213) that may be capable of acid–base chemistry. *Cys133 is not resolved in the WbmF crystal; its position here is inferred from the neighbouring residue (Gly132), which is visible in the electron density.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Comparison of active-site residues in WbmF, WbmG and WbmH compared with WbpP25 and GMER. (a) The SDR catalytic triad is conserved in WbmG and WbmH but not in WbmF where the residue normally found as serine or threonine superimposes onto the position of Ala131. (b) GMER is shown as an example of an SDR that catalyses 3,5-epimerisation of its substrate.21 The GMER epimerase catalytic residues Cys109 and His179 superimpose onto hydrophobic residues in WbmG and WbmH. WbmF has side chains in these positions (Cys133 and Asn213) that may be capable of acid–base chemistry. *Cys133 is not resolved in the WbmF crystal; its position here is inferred from the neighbouring residue (Gly132), which is visible in the electron density.
Mentions: SDR enzymes have a conserved catalytic triad that is involved in their oxidoreductase activity and that is classically composed of a spatially conserved serine, tyrosine and lysine (SYK) triad, for example, Ser142, Tyr166 and Lys170 in WbpP. The first member of this triad is sometimes found as a threonine (TYK),23 and the tyrosine can be replaced by a methionine (SMK).26 The SDR triad is conserved in WbmG and WbmH as TYK but in WbmF Ala131 superimposes onto the Ser/Thr position (Fig. 6a).

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