Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae

In this study, we investigated methionine synthase from Candida albicans (CaMET6p) and Saccharomyces cerevisiae (ScMET6p). We describe the cloning of CaMet6 and ScMet6, and the expression of both the enzymes in S. cerevisiae. CaMET6p is able to complement the disruption of met6 in S. cerevisiae. Following the purification of ScMET6p and CaMET6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET6p with CH3-H4PteGlu2, CH3-H4PteGlu3, CH3-H4PteGlu4, and l-homocysteine are 108, 84, 95, and 13 μM, respectively. The Michaelis constants for CaMET6p with CH3-H4PteGlu2, CH3-H4PteGlu3, CH3-H4PteGlu4, and l-homocysteine are 113, 129, 120, and 14 μM, respectively. Neither enzyme showed activity with CH3-H4PteGlu1 as a substrate. We conclude that ScMET6p and CaMET6p require a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to the bacterial metE homologs. The cloning, purification, and characterization of these enzymes lay the groundwork for inhibitor-design studies on the cobalamin-independent fungal methionine synthases.