Metabolic Engineering in Yeast Demonstrates ThatS-Adenosylmethionine Controls Flux through the Methylenetetrahydrofolate Reductase Reaction in Vivo

Abstract

One-carbon flux into methionine and S-adenosylmethionine (AdoMet) is thought to be controlled at the methylenetetrahydrofolate reductase (MTHFR) step. 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 this work, we used metabolic engineering in the yeast Saccharomyces cerevisiae to test this hypothesis. Like mammalian MTHFRs, the yeast MTHFR encoded by the MET13 gene is NADPH-dependent and is inhibited by AdoMet in vitro.ThiscontrastswithplantMTHFRs,whichareNADHdependent 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 domain and the AtMTHFR-1 C-terminal domain. Chimera-1 used both NADH and NADPH and was insensitive to AdoMet, supporting the view that the C-terminal domain is responsible for AdoMet inhibition. Engineered yeast expressing Chimera-1 accumulated 140-fold more AdoMet and 7-fold more methionine than did the wild-type and grew normally. Yeast expressing AtMTHFR-1 accumulated 8-fold more AdoMet. This is the first in vivo evidence that the AdoMet sensitivity and pyridine nucleotide preference of MTHFR control methylneogenesis.13 C labeling data indicated that glycine cleavage becomes a more prominent source of one-carbon units when Chimera-1 is expressed. Possibly related to this shift in one-carbon fluxes, total folate levels are doubled in yeast cells expressing Chimera-1.

The Folylpolyglutamate Synthetase Plastidial Isoform Is Required for Postembryonic Root Development in Arabidopsis

“…5-CHO-THF does not participate in C1 reactions, and its role in planta is not very well understood. In fact, this folate species has been shown to inhibit the activity of many folateutilizing enzymes (Stover and Schirch, 1993;Roje et al, 2002). Therefore, it was interesting to find that exogenous 5-CHO-THF rescued the root defects of atdfb Figure 10.…”

Regulation of S-Adenosylmethionine Levels in Saccharomyces cerevisiae

“…Formate-A yeast strain (SCY4) was previously engineered in which the coding sequence of Chimera-1 replaced the chromosomal MET13 gene in the wild type DAY4 strain (8). The growth rates of these two ser1 mutant strains in synthetic minimal medium supplemented with serine, leucine, histidine, tryptophan, and uracil were similar (doubling times, 2.5 Ϯ 0.2 h).…”

“…An excessive accumulation of CH 3 -THF may be detrimental to the de novo synthesis of nucleic acids that use non-methylated folates as substrates. A metabolically engineered yeast strain (SCY4) expressing an AdoMet-insensitive, chimeric yeast-plant MTHFR (Chimera-1) is able to accumulate greatly elevated pools of methionine and AdoMet (8). Although the folate pool is enlarged, the distribution of folate derivatives in SCY4 is not shifted toward the CH 3 -THF derivative (8).…”

“…A metabolically engineered yeast strain (SCY4) expressing an AdoMet-insensitive, chimeric yeast-plant MTHFR (Chimera-1) is able to accumulate greatly elevated pools of methionine and AdoMet (8). Although the folate pool is enlarged, the distribution of folate derivatives in SCY4 is not shifted toward the CH 3 -THF derivative (8). In the present study it is demonstrated that the hyperaccumulation of AdoMet is detrimental to yeast if the excess AdoMet cannot be sequestered in the vacuole using polyphosphate as a cationic exchanger.…”

“…The MTHFR encoded by MET13 has been shown to be NADPHdependent and inhibited by AdoMet in vivo (8). This physiolog- 1 The abbreviations used are: MTHFR, methylenetetrahydrofolate reductase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; THF, tetrahydrofolate; CH 2 -THF, 5,10-methylenetetrahydrofolate; CH 3 -THF, 5-methyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate; CH ϩ -THF, 5,10-methenyltetrahydrofolate; CBS, cystathionine ␤-synthase; GSH, glutathione, reduced form; HPLC, high performance liquid chromatography; CHES, 2-(cyclohexylamino)ethanesulfonic acid; kb, kilobase; EUROSCARF, European S. cerevisiae Archives for Functional Analysis.…”