title: Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae.
abstract: In this study, we investigated methionine synthase from Candida albicans (CaMET 6p) and Saccharomyces cerevisiae (ScMET 6p). We describe the cloning of CaMet 6 and ScMet 6, and the expression of both the enzymes in S. cerevisiae. CaMET 6p is able to complement the disruption of met 6 in S. cerevisiae. Following the purification of ScMET 6p and CaMET 6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET 6p 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 13 microM, respectively. The Michaelis constants for CaMET 6p 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 14 microM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) as a substrate. We conclude that ScMET 6p and CaMET 6p 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.
authors.affiliation: Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.
authors.forename: Huda S
authors.initials: HS
authors.lastname: Suliman
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Sawyer
authors.forename: Dean R
authors.initials: DR
authors.lastname: Appling
authors.forename: Jon D
authors.initials: JD
authors.lastname: Robertus
mesh_terms: D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D008780:Methyltransferases; C022725:5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase; C005984:5-methyltetrahydrofolate