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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) Silver stain analysis and (b) Western immunoblot of duplicate SDS-PAGE gels showing the LPS profiles of wild-type B. bronchiseptica (CN7635E) and CN7635E-derived mutants in wbmF (CNF0a), wbmG (CNG1a) and wbmH (CNH1d) and mutants carrying the complementation vectors for wbmF (pCompF), wbmG (pCompG) and wbmH (pCompH) or the empty vector (pCompEmpty). The positions of the B. bronchiseptica A-band (A) and B-band (B) species are indicated as well as the position of LPS that contains O antigen (O). The primary monoclonal antibody used in (b) that recognises O-band LPS was D13B11.35
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fig1: (a) Silver stain analysis and (b) Western immunoblot of duplicate SDS-PAGE gels showing the LPS profiles of wild-type B. bronchiseptica (CN7635E) and CN7635E-derived mutants in wbmF (CNF0a), wbmG (CNG1a) and wbmH (CNH1d) and mutants carrying the complementation vectors for wbmF (pCompF), wbmG (pCompG) and wbmH (pCompH) or the empty vector (pCompEmpty). The positions of the B. bronchiseptica A-band (A) and B-band (B) species are indicated as well as the position of LPS that contains O antigen (O). The primary monoclonal antibody used in (b) that recognises O-band LPS was D13B11.35

Mentions: To test the hypothesis that wbmF, wbmG and wbmH are involved in O-chain biosynthesis, each gene was disrupted by insertion of a tetracycline-resistance cassette. Mutation of the chromosomal genes was confirmed by Southern hybridisation (data not shown). The effects of these mutations were assessed by silver-stained SDS-PAGE analysis and immunoblotting of mutant LPS (Fig. 1). Mutation of wbmG or wbmH results in apparent abrogation of O-antigen synthesis, as the LPS from these mutants lacks O band as detected by either silver stain or Western blot. O-antigen expression in the wbmF mutant, CNF0a, is dramatically reduced compared with the wild type, but this strain retains its ability to express a small amount of material with the electrophoretic mobility of O-band LPS and which binds the O-antigen-specific monoclonal antibody, D13B11.


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) Silver stain analysis and (b) Western immunoblot of duplicate SDS-PAGE gels showing the LPS profiles of wild-type B. bronchiseptica (CN7635E) and CN7635E-derived mutants in wbmF (CNF0a), wbmG (CNG1a) and wbmH (CNH1d) and mutants carrying the complementation vectors for wbmF (pCompF), wbmG (pCompG) and wbmH (pCompH) or the empty vector (pCompEmpty). The positions of the B. bronchiseptica A-band (A) and B-band (B) species are indicated as well as the position of LPS that contains O antigen (O). The primary monoclonal antibody used in (b) that recognises O-band LPS was D13B11.35
© Copyright Policy
Related In: Results  -  Collection

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

fig1: (a) Silver stain analysis and (b) Western immunoblot of duplicate SDS-PAGE gels showing the LPS profiles of wild-type B. bronchiseptica (CN7635E) and CN7635E-derived mutants in wbmF (CNF0a), wbmG (CNG1a) and wbmH (CNH1d) and mutants carrying the complementation vectors for wbmF (pCompF), wbmG (pCompG) and wbmH (pCompH) or the empty vector (pCompEmpty). The positions of the B. bronchiseptica A-band (A) and B-band (B) species are indicated as well as the position of LPS that contains O antigen (O). The primary monoclonal antibody used in (b) that recognises O-band LPS was D13B11.35
Mentions: To test the hypothesis that wbmF, wbmG and wbmH are involved in O-chain biosynthesis, each gene was disrupted by insertion of a tetracycline-resistance cassette. Mutation of the chromosomal genes was confirmed by Southern hybridisation (data not shown). The effects of these mutations were assessed by silver-stained SDS-PAGE analysis and immunoblotting of mutant LPS (Fig. 1). Mutation of wbmG or wbmH results in apparent abrogation of O-antigen synthesis, as the LPS from these mutants lacks O band as detected by either silver stain or Western blot. O-antigen expression in the wbmF mutant, CNF0a, is dramatically reduced compared with the wild type, but this strain retains its ability to express a small amount of material with the electrophoretic mobility of O-band LPS and which binds the O-antigen-specific monoclonal antibody, D13B11.

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