The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis.
MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged 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 dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that 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.
Department of Molecular Genetics, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.
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Raux
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McVeigh
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Peters
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Leustek
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Warren
D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005294:Ferrochelatase; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006418:Heme / Q000031:analogs & derivatives* / Q000096:biosynthesis; D008190:Lyases / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012486:Salmonella typhimurium / Q000235:genetics; D014805:Vitamin B 12 / Q000096:biosynthesis*
D001426:Bacterial Proteins; D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; C009272:siroheme; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase; D008190:Lyases; C103747:MET8 protein, S cerevisiae; C033211:cobaltochelatase; D005294:Ferrochelatase; D014805:Vitamin B 12