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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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Nucleotide binding in WbmF, WbmG and WbmH. Proteins are oriented with the Rossmann domains in the lower part of each structure. Cartoons are used to represent protein backbone; bound nucleotides are shown as ball-and-stick and key protein side chains as sticks. The boxed panels show details of UMP-binding pockets in (a) His6-WbmF, UDP co-crystal, (b) His6-WbmG crystal soaked with UDP-glucose and (c) His6-WbmH. In (a) and (b), UMP is shown as sticks, and spheres represent atoms within 3.5 Å of the bound nucleotide. Probable hydrogen-bonding interactions are shown as dashed lines. In (c) spheres represent ordered water molecules in this binding pocket. Carbon atoms are coloured rainbow for protein, white for bound nucleotides; oxygen atoms are coloured red; nitrogen is blue and phosphorus orange.
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fig3: Nucleotide binding in WbmF, WbmG and WbmH. Proteins are oriented with the Rossmann domains in the lower part of each structure. Cartoons are used to represent protein backbone; bound nucleotides are shown as ball-and-stick and key protein side chains as sticks. The boxed panels show details of UMP-binding pockets in (a) His6-WbmF, UDP co-crystal, (b) His6-WbmG crystal soaked with UDP-glucose and (c) His6-WbmH. In (a) and (b), UMP is shown as sticks, and spheres represent atoms within 3.5 Å of the bound nucleotide. Probable hydrogen-bonding interactions are shown as dashed lines. In (c) spheres represent ordered water molecules in this binding pocket. Carbon atoms are coloured rainbow for protein, white for bound nucleotides; oxygen atoms are coloured red; nitrogen is blue and phosphorus orange.

Mentions: These proteins all exhibit the typical SDR family architecture (Fig. 3). The structures each comprise two domains. The first of these is the N-terminal Rossmann fold domain in which a central β sheet is flanked by two layers of α helices. The cofactor-binding motif GxxGxxG is located at the C-terminal edge of this β sheet, which has seven parallel β strands running in the order 3, 2, 1, 4, 5, 6, 7. The second domain is largely made up of C-terminal sequence and contains all of the residues involved in binding of the nucleotide portion of the substrate. The catalytic sites in SDR enzymes are located at the interface of these two domains where, in sugar-nucleotide-modifying enzymes, the substrate hexose is brought into proximity with the cofactor nicotinamide ring. The main architectural differences between these three proteins lie in their C-terminal regions (Fig. 3). The C terminus of His6-WbmG consists of a loop that stretches out from the C-terminal domain to interact with the Rossmann domain. In His6-WbmH, this loop is not visible in the density, although this difference may not reflect any distinction between the real structures of the two proteins in solution. In WbmF, the last 30 residues form a large bent helix that covers two faces of the C-terminal domain. There is also an insertion of 14 amino acids in WbmF relative to the other structures, including all residues from Gly198 to Arg212. This extra loop extends over the nicotinamide end of the cofactor-binding pocket.


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)

Nucleotide binding in WbmF, WbmG and WbmH. Proteins are oriented with the Rossmann domains in the lower part of each structure. Cartoons are used to represent protein backbone; bound nucleotides are shown as ball-and-stick and key protein side chains as sticks. The boxed panels show details of UMP-binding pockets in (a) His6-WbmF, UDP co-crystal, (b) His6-WbmG crystal soaked with UDP-glucose and (c) His6-WbmH. In (a) and (b), UMP is shown as sticks, and spheres represent atoms within 3.5 Å of the bound nucleotide. Probable hydrogen-bonding interactions are shown as dashed lines. In (c) spheres represent ordered water molecules in this binding pocket. Carbon atoms are coloured rainbow for protein, white for bound nucleotides; oxygen atoms are coloured red; nitrogen is blue and phosphorus orange.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Nucleotide binding in WbmF, WbmG and WbmH. Proteins are oriented with the Rossmann domains in the lower part of each structure. Cartoons are used to represent protein backbone; bound nucleotides are shown as ball-and-stick and key protein side chains as sticks. The boxed panels show details of UMP-binding pockets in (a) His6-WbmF, UDP co-crystal, (b) His6-WbmG crystal soaked with UDP-glucose and (c) His6-WbmH. In (a) and (b), UMP is shown as sticks, and spheres represent atoms within 3.5 Å of the bound nucleotide. Probable hydrogen-bonding interactions are shown as dashed lines. In (c) spheres represent ordered water molecules in this binding pocket. Carbon atoms are coloured rainbow for protein, white for bound nucleotides; oxygen atoms are coloured red; nitrogen is blue and phosphorus orange.
Mentions: These proteins all exhibit the typical SDR family architecture (Fig. 3). The structures each comprise two domains. The first of these is the N-terminal Rossmann fold domain in which a central β sheet is flanked by two layers of α helices. The cofactor-binding motif GxxGxxG is located at the C-terminal edge of this β sheet, which has seven parallel β strands running in the order 3, 2, 1, 4, 5, 6, 7. The second domain is largely made up of C-terminal sequence and contains all of the residues involved in binding of the nucleotide portion of the substrate. The catalytic sites in SDR enzymes are located at the interface of these two domains where, in sugar-nucleotide-modifying enzymes, the substrate hexose is brought into proximity with the cofactor nicotinamide ring. The main architectural differences between these three proteins lie in their C-terminal regions (Fig. 3). The C terminus of His6-WbmG consists of a loop that stretches out from the C-terminal domain to interact with the Rossmann domain. In His6-WbmH, this loop is not visible in the density, although this difference may not reflect any distinction between the real structures of the two proteins in solution. In WbmF, the last 30 residues form a large bent helix that covers two faces of the C-terminal domain. There is also an insertion of 14 amino acids in WbmF relative to the other structures, including all residues from Gly198 to Arg212. This extra loop extends over the nicotinamide end of the cofactor-binding pocket.

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