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

Arch Biochem Biophys. 2005 Sep 1;441(1):56-63. doi: 10.1016/j.abb.2005.06.016.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Candida albicans / enzymology*
  • Candida albicans / genetics
  • Enzyme Activation
  • Kinetics
  • Methyltransferases / chemistry*
  • Methyltransferases / genetics
  • Methyltransferases / isolation & purification*
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Tetrahydrofolates / chemistry*

Substances

  • Recombinant Proteins
  • Tetrahydrofolates
  • Methyltransferases
  • 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase
  • 5-methyltetrahydrofolate