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

Raux-Deery, Evelyne and McVeigh, Treasa and Peters, Sarah E. and Leustek, Thomas and Warren, Martin J. (1999) The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis. Biochemical Journal , 338 (3). pp. 701-8. ISSN 0264-6021 . (The full text of this publication is not available from this repository)

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Abstract

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.

Item Type: Article
Additional information:
Uncontrolled keywords: *Bacterial Proteins Base Sequence Cloning, Molecular DNA Primers Escherichia coli/genetics *Ferrochelatase Fungal Proteins/genetics/*metabolism Genetic Complementation Test Heme/*analogs & derivatives/biosynthesis Lyases/metabolism Methyltransferases/metabolism Recombinant Proteins/genetics/metabolism Saccharomyces cerevisiae/*metabolism *Saccharomyces cerevisiae Proteins Salmonella typhimurium/genetics Vitamin B 12/*biosynthesis
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: Martin Warren
Date Deposited: 15 Oct 2009 08:10
Last Modified: 23 Jun 2014 15:51
Resource URI: http://kar.kent.ac.uk/id/eprint/11074 (The current URI for this page, for reference purposes)
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