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Literature summary extracted from

  • Sah, S.; Lahry, K.; Talwar, C.; Singh, S.; Varshney, U.
    Monomeric NADH-oxidizing methylenetetrahydrofolate reductases from Mycobacterium smegmatis lack flavin coenzyme (2020), J. Bacteriol., 202, e00709-19 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.15 gene MSMEG_6596, DNA and amino acid sequence determination and analysis, sequence comparisons Mycolicibacterium smegmatis
1.5.1.15 gene MSMEG_6649, DNA and amino acid sequence determination and analysis, sequence comparisons Mycolicibacterium smegmatis

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.15 additional information construction of the Mycobacterium smegmatis DELTAmsmeg_6596 strain. The mutant is partially auxotrophic for methionine and grows only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the DELTAmsmeg_6596 strain is more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim) Mycolicibacterium smegmatis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.15 0.0325
-
 NADH pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 0.0632
-
 5,10-methenyltetrahydrofolate pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 0.108
-
 NADH pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 0.145
-
 5,10-methenyltetrahydrofolate pH 7.6, 22°C Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.15 5,10-methenyltetrahydrofolate + NADH + H+ Mycolicibacterium smegmatis
-
 5,10-methylenetetrahydrofolate + NAD+
-
 ir
1.5.1.15 5,10-methenyltetrahydrofolate + NADH + H+ Mycolicibacterium smegmatis ATCC 700084
-
 5,10-methylenetetrahydrofolate + NAD+
-
 ir

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.15 Mycolicibacterium smegmatis A0R6M0
-
-
1.5.1.15 Mycolicibacterium smegmatis A0R6S0
-
-
1.5.1.15 Mycolicibacterium smegmatis ATCC 700084 A0R6M0
-
-
1.5.1.15 Mycolicibacterium smegmatis ATCC 700084 A0R6S0
-
-
1.5.1.15 no activity by Mycobacterium smegmatis MSMEG_6664
-
-
-
1.5.1.15 no activity by Mycobacterium smegmatis MSMEI_6484
-
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.15 5,10-methenyltetrahydrofolate + NADH + H+
-
 Mycolicibacterium smegmatis 5,10-methylenetetrahydrofolate + NAD+
-
 ir
1.5.1.15 5,10-methenyltetrahydrofolate + NADH + H+
-
 Mycolicibacterium smegmatis ATCC 700084 5,10-methylenetetrahydrofolate + NAD+
-
 ir
1.5.1.15 additional information identification of the reaction product of MSMEG_6596 by LC-MS/MS Mycolicibacterium smegmatis ?
-
-
1.5.1.15 additional information identification of the reaction product of MSMEG_6649 by LC-MS/MS Mycolicibacterium smegmatis ?
-
-
1.5.1.15 additional information identification of the reaction product of MSMEG_6596 by LC-MS/MS Mycolicibacterium smegmatis ATCC 700084 ?
-
-
1.5.1.15 additional information identification of the reaction product of MSMEG_6649 by LC-MS/MS Mycolicibacterium smegmatis ATCC 700084 ?
-
-

Subunits

EC Number Subunits Comment Organism
1.5.1.15 monomer MSMEG_6596 is a monomeric protein and lacks flavin coenzyme Mycolicibacterium smegmatis
1.5.1.15 monomer MSMEG_6649 is a monomeric protein and lacks flavin coenzyme Mycolicibacterium smegmatis

Synonyms

EC Number Synonyms Comment Organism
1.5.1.15 MSMEG_6596
-
 Mycolicibacterium smegmatis
1.5.1.15 MSMEG_6649
-
 Mycolicibacterium smegmatis
1.5.1.15 MTHFR
-
 Mycolicibacterium smegmatis
1.5.1.15 MTHFR1
-
 Mycolicibacterium smegmatis
1.5.1.15 MTHFR2
-
 Mycolicibacterium smegmatis
1.5.1.15 NADH-oxidizing methylenetetrahydrofolate reductase
-
 Mycolicibacterium smegmatis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.15 22
-
 assay at room temperature Mycolicibacterium smegmatis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.15 0.688
-
 5,10-methenyltetrahydrofolate pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 1.322
-
 NADH pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 4.063
-
 5,10-methenyltetrahydrofolate pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 6.095
-
 NADH pH 7.6, 22°C Mycolicibacterium smegmatis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.15 7.6
-
 assay at Mycolicibacterium smegmatis

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.15 additional information MSMEG_6596 is a monomeric protein and lacks flavin coenzyme. No activity with NADP(H) Mycolicibacterium smegmatis
1.5.1.15 additional information MSMEG_6649 is a monomeric protein and lacks flavin coenzyme. No activity with NADP(H) Mycolicibacterium smegmatis
1.5.1.15 NAD+
-
 Mycolicibacterium smegmatis
1.5.1.15 NADH
-
 Mycolicibacterium smegmatis

General Information

EC Number General Information Comment Organism
1.5.1.15 malfunction the Mycobacterium smegmatis DELTAmsmeg_6596 strain is partially auxotrophic for methionine and grows only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the DELTAmsmeg_6596 strain is more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim) Mycolicibacterium smegmatis
1.5.1.15 physiological function MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. In Mycobacterium smegmatis, the catalytic efficiency (kcat/Km) in vitro of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH is 13.5 and 15.3fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation is further supported by better rescue of the Escherichia coli DELTAmthfr mutant strain by MTHFR1 than by MTHFR2. The isozymes 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. MTHFR activity is required during stress from folate pathway inhibitors. The expression of MSMEG_6596 restores maximum resistance or growth, followed by MSMEG_6649, providing antifolate resistance to Mycobacterium smegmatis Mycolicibacterium smegmatis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.1.15 4.75
-
 5,10-methenyltetrahydrofolate pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 12.24
-
 NADH pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 64.29
-
 5,10-methenyltetrahydrofolate pH 7.6, 22°C Mycolicibacterium smegmatis
1.5.1.15 94.68
-
 NADH pH 7.6, 22°C Mycolicibacterium smegmatis