The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described. MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis. The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids. The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids. The deduced amino acid sequences of these two genes are 34% identical to each other and 32-37% identical to the human MTHFR. A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy. Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy. Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine. Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine. The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used. Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain. In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells. Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes.

• PMID: 10600168
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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Raymond RK, Kastanos EK, Appling DR

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