The gene for cobalamin-independent methionine synthase is essential in Candida albicans: A potential antifungal target

Methionine synthase catalyzes the transfer of a methyl group from tetrahydrofolate to homocysteine to produce methionine. Although mammalian enzymes are cobalamin-dependent, fungal methionine synthases are cobalamin-independent. The opportunistic pathogen Candida albicans is a diploid and carries two copies of the methionine synthase gene, MET6. Homologous recombination was used to disrupt a single MET6 gene. MET6/met6 knock-outs, deleted with either the URA3 or ARG4 cassette, grew as well as the wild-type strain. However, we were unable to obtain a viable met6/met6 deletion strain, even on media supplemented with exogenous methionine. This suggests that methionine synthase is essential to C. albicans. To explore this further, a C. albicans strain was constructed in which one MET6 locus was deleted and the second placed under a regulatable promoter. The conditional mutant grew well under inducing conditions, even in the absence of methionine. It would not grow under repressing conditions in the absence of methionine, but would grow when the media was supplemented with exogenous methionine. A Western blot showed that a small amount of enzyme was expressed under repressing conditions. Taken together, these data reveal that methionine is necessary for growth of C. albicans, but not sufficient—a minimal level of methionine synthase expression is required, perhaps to limit homocysteine toxicity. Furthermore, these results suggest that cobalamin-independent methionine synthase is a plausible target for the design of antifungal agents.