Identification and characterization of GDP-D-mannose 4,6-dehydratase and GDP-L-fucose synthetase 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 muM 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 muM 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 off. 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. (C) 2001 Academic Press.