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 CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 108, 84, 95, and 13muM, respectively. The Michaelis constants for CaMET6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 113, 129, 120, and 14muM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) 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.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|