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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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Pi-stacking interactions in binding of uracil or thymine. Amino acids that directly interact with the substrate nucleobase are shown for B. bronchiseptica WbmF and WbmG, E. coli GalE, S. venezuelae DesIV and P. aeruginosa WbpP with carbon atoms coloured yellow. UMP or dTDP from each structure is shown with carbon atoms in white. Amino acid side chains that have pi-stacking interactions with the base are labelled, and hydrogen-bonding interactions with the peptide backbone are indicated as dashed lines. Images for GalE, DesIV and WbpP were prepared using PDB files 1LRL,241R6D23 and 1SB8,25 respectively.
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fig5: Pi-stacking interactions in binding of uracil or thymine. Amino acids that directly interact with the substrate nucleobase are shown for B. bronchiseptica WbmF and WbmG, E. coli GalE, S. venezuelae DesIV and P. aeruginosa WbpP with carbon atoms coloured yellow. UMP or dTDP from each structure is shown with carbon atoms in white. Amino acid side chains that have pi-stacking interactions with the base are labelled, and hydrogen-bonding interactions with the peptide backbone are indicated as dashed lines. Images for GalE, DesIV and WbpP were prepared using PDB files 1LRL,241R6D23 and 1SB8,25 respectively.

Mentions: One common feature of substrate binding in SDR enzymes is pi stacking of the nucleobase with an amino acid side chain. In WbmF, this interaction involves Glu231 (Fig. 3a). Unusually, in WbmG the pi-stacking residue (Phe190) is the one that hydrogen bonds with the uracil N-3 (Fig. 3b). In WbmF, E. coli UDP-galactose 4-epimerase (GalE),24S. venezuelae DesIV,23 and Pseudomonas aeruginosa WbpP,25 the hydrophobic contact is provided by the residue in the n + 2 position, whose backbone amide hydrogen bonds with the base's O2 group (Fig. 5). This unusual feature of the substrate binding in WbmG could not have been predicted from sequence alignments alone.


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)

Pi-stacking interactions in binding of uracil or thymine. Amino acids that directly interact with the substrate nucleobase are shown for B. bronchiseptica WbmF and WbmG, E. coli GalE, S. venezuelae DesIV and P. aeruginosa WbpP with carbon atoms coloured yellow. UMP or dTDP from each structure is shown with carbon atoms in white. Amino acid side chains that have pi-stacking interactions with the base are labelled, and hydrogen-bonding interactions with the peptide backbone are indicated as dashed lines. Images for GalE, DesIV and WbpP were prepared using PDB files 1LRL,241R6D23 and 1SB8,25 respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Pi-stacking interactions in binding of uracil or thymine. Amino acids that directly interact with the substrate nucleobase are shown for B. bronchiseptica WbmF and WbmG, E. coli GalE, S. venezuelae DesIV and P. aeruginosa WbpP with carbon atoms coloured yellow. UMP or dTDP from each structure is shown with carbon atoms in white. Amino acid side chains that have pi-stacking interactions with the base are labelled, and hydrogen-bonding interactions with the peptide backbone are indicated as dashed lines. Images for GalE, DesIV and WbpP were prepared using PDB files 1LRL,241R6D23 and 1SB8,25 respectively.
Mentions: One common feature of substrate binding in SDR enzymes is pi stacking of the nucleobase with an amino acid side chain. In WbmF, this interaction involves Glu231 (Fig. 3a). Unusually, in WbmG the pi-stacking residue (Phe190) is the one that hydrogen bonds with the uracil N-3 (Fig. 3b). In WbmF, E. coli UDP-galactose 4-epimerase (GalE),24S. venezuelae DesIV,23 and Pseudomonas aeruginosa WbpP,25 the hydrophobic contact is provided by the residue in the n + 2 position, whose backbone amide hydrogen bonds with the base's O2 group (Fig. 5). This unusual feature of the substrate binding in WbmG could not have been predicted from sequence alignments alone.

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
Related in: MedlinePlus