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 de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFR. 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. In vitro, the catalytic efficiency (kcat/Km) of MSMEG_6596 (MTHFR1) for 5,10-CH2-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 E. coliΔmthfr by MTHFR1 than MTHFR2. As identified by LC-MS/MS, the product of MTHFR1 or MTHFR2 catalysed reactions was 5-CH3-THF. M. smegmatisΔmsmeg_6596 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 Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins which are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria. Importance 5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR failed to reveal their function as MTHFR. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649 as MTHFR1 and MTHFR2, respectively. Interestingly, both the MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (SCP, PAS, SMX and TMP). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.