Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole
enzyme responsible for generation of
5-methyltetrahydrofolate, which is required for
methionine synthesis and provision of methyl groups via
S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct
methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant
enzyme was produced in Escherichia coli. MTHFR was not inhibited by
S-adenosylmethionine and, uniquely among
folate-metabolizing
enzymes, showed dual-cofactor specificity with
NADH and
NADPH under physiological conditions. MTHFR null mutants (mthfr(-)) lacked
5-methyltetrahydrofolate, the most abundant intracellular
folate, and could not utilize exogenous
homocysteine for growth. Under conditions of
methionine limitation mthfr(-) mutant cells grew poorly, whereas their growth was normal in standard
culture media. Neither in vitro MTHFR activity nor the growth of mthfr(-) mutants or MTHFR overexpressors were differentially affected by
antifolates known to inhibit parasite growth via targets beyond
dihydrofolate reductase and
pteridine reductase 1. In a mouse model of
infection mthfr(-) mutants showed good infectivity and virulence, indicating that sufficient
methionine is available within the parasitophorous vacuole to meet the needs of the parasite.