Isolation, characterization, and functional expression of cDNAs encoding NADH-dependent methylenetetrahydrofolate reductase from higher plants.
Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants. Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms. These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells. Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences. Southern analyses and genomic evidence indicate that Arabidopsis has two MTHFR genes and that maize has at least two. A carboxyl-terminal polyhistidine tag was added to one Arabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity. Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of approximately 66-kDa subunits. Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited by S-adenosylmethionine. An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10(-3). Consistent with this, leaf tissues metabolized [methyl-(14)C]methyltetrahydrofolate to serine, sugars, and starch. A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate.
Roje
S
S
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.
Wang
H
H
McNeil
S D
SD
Raymond
R K
RK
Appling
D R
DR
Shachar-Hill
Y
Y
Bohnert
H J
HJ
Hanson
A D
AD
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis
D018076:DNA, Complementary; D010940:Plant Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)