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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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The UDP-binding pockets are shown for (a) His6-WbmF, soaked with UDP and (b) the His6-WbmG, UDP co-crystal. Spheres represent atoms within 3.5 Å of the bound UDP; NAD indicates the nicotinamide ring of the NAD cofactor. (c) dTDP-glucose bound in the active site of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase DesIV from S. venezuelae (PDB ID 1R6D).23 The substrate-binding pockets are all shown from the same angle to enable comparison of the relative positions of the UDP diphosphates in (a) and (b) with the phosphates in dTDP-glucose in (c).
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fig4: The UDP-binding pockets are shown for (a) His6-WbmF, soaked with UDP and (b) the His6-WbmG, UDP co-crystal. Spheres represent atoms within 3.5 Å of the bound UDP; NAD indicates the nicotinamide ring of the NAD cofactor. (c) dTDP-glucose bound in the active site of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase DesIV from S. venezuelae (PDB ID 1R6D).23 The substrate-binding pockets are all shown from the same angle to enable comparison of the relative positions of the UDP diphosphates in (a) and (b) with the phosphates in dTDP-glucose in (c).

Mentions: Both α and β nucleotide phosphates could be modelled in the data from a UDP soak into a WbmF-NAD+ co-crystal and co-crystallisation of His6-WbmG with UDP. In both structures the β-phosphate is coordinated by a conserved arginine (Fig. 4a and b), although the precise location of this phosphate is different in the two structures and both are inconsistent with the β-phosphate positions of bound substrate in the structure of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase, DesIV, from S. venezuelae (Fig. 4c).23 In this DesIV structure [Protein Data Bank (PDB) ID 1R6D] the position of the substrate is consistent with the necessary overlap of molecular orbitals required for hydride transfer from the glucose C-4 to the cofactor. It is likely, therefore, that the β-phosphate positions in WbmF and WbmG structures do not represent the positions of equivalent phosphates when the native substrates for these enzymes are bound in the active site.


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)

The UDP-binding pockets are shown for (a) His6-WbmF, soaked with UDP and (b) the His6-WbmG, UDP co-crystal. Spheres represent atoms within 3.5 Å of the bound UDP; NAD indicates the nicotinamide ring of the NAD cofactor. (c) dTDP-glucose bound in the active site of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase DesIV from S. venezuelae (PDB ID 1R6D).23 The substrate-binding pockets are all shown from the same angle to enable comparison of the relative positions of the UDP diphosphates in (a) and (b) with the phosphates in dTDP-glucose in (c).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: The UDP-binding pockets are shown for (a) His6-WbmF, soaked with UDP and (b) the His6-WbmG, UDP co-crystal. Spheres represent atoms within 3.5 Å of the bound UDP; NAD indicates the nicotinamide ring of the NAD cofactor. (c) dTDP-glucose bound in the active site of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase DesIV from S. venezuelae (PDB ID 1R6D).23 The substrate-binding pockets are all shown from the same angle to enable comparison of the relative positions of the UDP diphosphates in (a) and (b) with the phosphates in dTDP-glucose in (c).
Mentions: Both α and β nucleotide phosphates could be modelled in the data from a UDP soak into a WbmF-NAD+ co-crystal and co-crystallisation of His6-WbmG with UDP. In both structures the β-phosphate is coordinated by a conserved arginine (Fig. 4a and b), although the precise location of this phosphate is different in the two structures and both are inconsistent with the β-phosphate positions of bound substrate in the structure of a D128N, E129Q mutant of dTDP-glucose 4,6-dehydratase, DesIV, from S. venezuelae (Fig. 4c).23 In this DesIV structure [Protein Data Bank (PDB) ID 1R6D] the position of the substrate is consistent with the necessary overlap of molecular orbitals required for hydride transfer from the glucose C-4 to the cofactor. It is likely, therefore, that the β-phosphate positions in WbmF and WbmG structures do not represent the positions of equivalent phosphates when the native substrates for these enzymes are bound in the active site.

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