Identification and characterization of GDP-d-mannose 4,6-dehydratase and GDP-l-fucose snthetase in a GDP-l-fucose biosynthetic gene cluster from Helicobacter pylori.
In this study two open reading frames, namely HP0044 and HP0045 from H. pylori, were cloned and overexpressed in E. coli. The two recombinant proteins were demonstrated to have GDP-d-mannose 4,6-dehydratase (GMD) and GDP-l-fucose synthetase (GFS) activities, respectively. The recombinant GMD was a tetramer and had an optimum pH of 6.5. Exogenous NADP(+) was essential for its activity. The K(m) and K(cat) for GDP-d-mannose were 117.3 microM and 0.27 s(-1), respectively. The recombinant GFS was a homodimer with an optimum pH of 8.0. The K(m) and K(cat) for GDP-4-keto-6-deoxy-d-mannose were 64.08 microM and 0.75 s(-1), respectively. It can use both NADPH and NADH, but less efficient with the latter. Amino acid sequence alignment and phylogenetic analysis showed that H. pylori GFS was highly homologous to the GFS of E. coli O111 and both of them were located on a separate phylogenetic branch from other GFS. The unique clustering and origin of the two genes were also discussed.
Wu
B
B
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Zhang
Y
Y
Wang
P G
PG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D016384:Consensus Sequence; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D006154:Guanosine Diphosphate Fucose / Q000096:biosynthesis*; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D005810:Multigene Family; D019906:Nuclear Magnetic Resonance, Biomolecular; D010802:Phylogeny; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D046911:Macromolecular Substances; D021122:Protein Subunits; D011994:Recombinant Proteins; D006154:Guanosine Diphosphate Fucose; C023078:GDPfucose synthetase; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase