The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Sa lmonella typhimurium cysG, indicating that the genes are involved in the tr ansformation of uroporphyrinogen III into sirohaem. In the present study, w e have demonstrated complementation of defined cysG mutants of Sal. typhimu rium and Escherichia coli, with either MET1 or MET8 cloned in tandem with P seudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-L-methionine uroporphyrinogen III trans methylase activity, and MET8 encodes the dehydrogenase and chelatase activi ties (all three functions are encoded by Sal. typhimurium and E. coli cysG) . MET8 was further cloned into pET14b to allow expression of the protein wi th an N-terminal His-tag. After purification, the functions of the His-tagg ed Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD(+) and Co2+, The results demonstrated that Met8p acts as a dehydrogena se and chelatase in the biosynthesis of sirohaem. Moreover, despite the fac t that S. cerevisiae does not make cobalamins de novo, we have shown also t hat MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference In the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.