title: Metabolic engineering in yeast demonstrates that S-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. This contrasts with plant MTHFRs, which 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 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.
authors.affiliation: Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Sanja
authors.initials: S
authors.lastname: Roje
authors.forename: Sherwin Y
authors.initials: SY
authors.lastname: Chan
authors.forename: Fatma
authors.initials: F
authors.lastname: Kaplan
authors.forename: Rhonda K
authors.initials: RK
authors.lastname: Raymond
authors.forename: Donald W
authors.initials: DW
authors.lastname: Horne
authors.forename: Dean R
authors.initials: DR
authors.lastname: Appling
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005492:Folic Acid / Q000378:metabolism; D005816:Genetic Complementation Test; D007700:Kinetics; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D009249:NADP; D012436:S-Adenosylmethionine; D005492:Folic Acid; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)