Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases from Mycobacterium smegmatis Lack Flavin Coenzyme.

5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for biosynthesis of methionine. Our and analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in , failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. , the catalytic efficiency (/ ) of MSMEG_6596 (MTHFR1) for 5,10-CH-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the Δ strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH-THF. The Δ strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δ strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, -aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in methionine biosynthesis and required for antifolate resistance in mycobacteria. MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our and analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. deleted for the major () was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, -aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in methionine biosynthesis and required for antifolate resistance in mycobacteria.