Monomeric NADH-oxidizing methylenetetrahydrofolate reductases from mycobacterium smegmatis lack flavin coenzyme

Sah, S and Lahry, K and Talwar, C and Singh, S and Varshney, U (2020) Monomeric NADH-oxidizing methylenetetrahydrofolate reductases from mycobacterium smegmatis lack flavin coenzyme. In: Journal of Bacteriology, 202 (12).

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Abstract

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 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. 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 the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 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 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 that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.

Item Type:	Journal Article
Publication:	Journal of Bacteriology
Publisher:	American Society for Microbiology
Additional Information:	The copyright for this article belongs to American Society for Microbiology.
Keywords:	5,10 methylenetetrahydrofolate reductase (FADH2); aminosalicylic acid; bacterial enzyme; flavine adenine nucleotide; flavine mononucleotide; flavoprotein; glycine hydroxymethyltransferase; homocysteine methyltransferase; methionine; methionine synthase; methylenetetrahydrofolate dehydrogenase; methylenetetrahydrofolate reductase 1; methylenetetrahydrofolate reductase 2; reduced nicotinamide adenine dinucleotide; RNA polymerase; sulfachlorpyridazine; sulfamethoxazole; trimethoprim; unclassified drug; bacterial protein; methylenetetrahydrofolate reductase (NADPH2); nicotinamide adenine dinucleotide; riboflavin derivative; 5,10 methylenetetrahydrofolate reductase (FADH2); flavine nucleotide; folic acid; folic acid antagonist; quercetin; reduced nicotinamide adenine dinucleotide; sulfachlorpyridazine; sulfamethoxazole; trimethoprim, Acetobacterium woodii; amino terminal sequence; Article; bacterial growth; bacterial strain; coenzyme; controlled study; enzyme activity; enzyme mechanism; Escherichia coli; in vitro study; in vivo study; liquid chromatography-mass spectrometry; mass spectrometry; metabolism; Moorella thermoacetica; Mycobacterium smegmatis; nonhuman; open reading frame; priority journal; Salmonella enterica serovar Typhimurium; sequence alignment; Thermus thermophilus; amino acid sequence; chemistry; coenzyme; enzymology; genetics; kinetics; metabolism; Mycobacterium smegmatis; sequence homology; catalytic efficiency; Mycobacterium tuberculosis, Amino Acid Sequence; Bacterial Proteins; Coenzymes; Flavins; Kinetics; Methylenetetrahydrofolate Reductase (NADPH2); Mycobacterium smegmatis; NAD; Sequence Homology, Amino Acid
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	06 Feb 2023 07:21
Last Modified:	06 Feb 2023 07:21
URI:	https://eprints.iisc.ac.in/id/eprint/79880

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