Function and regulation of two methylenetetrahydrofolate reductase isozymes in Saccharomyces cerevisiae

Abstract

One-carbon flux into methionine and S-adenosylmethionine (AdoMet) is thought to be controlled by the methylenetetrahydrofolate reductase (MTHFR) reaction. MTHFR catalyzes the physiologically irreversible reduction of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate. Mammalian MTHFRs are inhibited by AdoMet in vitro, and it has been proposed that methyl group biogenesis is regulated in vivo by this feedback loop. In the present study, the yeast Saccharomyces cerevisiae was metabolically engineered to examine this hypothesis. Analogous to mammalian MTHFRs, the yeast MTHFR encoded by the MET13 gene is NADPH-dependent and is inhibited by AdoMet in vitro. In contrast, the plant MTHFRs are NADH-dependent and AdoMet-insensitive. To manipulate flux through the MTHFR reaction in yeast, the chromosomal copy of MET13 was replaced by an Arabidopsis MTHFR cDNA (AtMTHFR-1), or by a chimeric sequence (CHIMERA-1) comprising the yeast N-terminal catalytic domain and the AtMTHFR-1 C-terminal domain. Chimera-1 used both NADH and NADPH and was insensitive to AdoMet. Engineered yeast expressing Chimera-1 accumulated 140-fold more AdoMet and seven-fold more methionine than the wild type, and grew normally. This is the first in vivo evidence that the AdoMet sensitivity and pyridine nucleotide preference of MTHFR control methylneogenesis. The ability of yeast to hyperaccumulate AdoMet with no appreciable growth defect was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation. Previous studies have established that AdoMet is distributed between the cytosol and the vacuole. A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation. The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate, but did not occur when these supplements were replaced by serine. The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to sulfur metabolism. In addition, S. cerevisiae possesses a highly conserved homolog to MET13, encoded by the MET12 gene. The deduced amino acid sequence of MET12 shares 37% identity to Met13p and AtMTHFR-1, and is 31% identical to the human MTHFR. Studies into the function and regulation of the Met12p isozyme are described.