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Title: Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

Abstract

Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.

Authors:
;  [1];  [1]
  1. Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas, Austin, TX 78712 (United States)
Publication Date:
OSTI Identifier:
22199689
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 382; Journal Issue: 4; Other Information: Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ARABIDOPSIS; CANDIDA; DESIGN; ENZYME ACTIVITY; ENZYMES; METHIONINE; MOLECULAR MODELS; MUTANTS; THREE-DIMENSIONAL CALCULATIONS

Citation Formats

Prasannan, Priya, Suliman, Huda S., and Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans. United States: N. p., 2009. Web. doi:10.1016/J.BBRC.2009.03.098.
Prasannan, Priya, Suliman, Huda S., & Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans. United States. doi:10.1016/J.BBRC.2009.03.098.
Prasannan, Priya, Suliman, Huda S., and Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu. Fri . "Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans". United States. doi:10.1016/J.BBRC.2009.03.098.
@article{osti_22199689,
title = {Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans},
author = {Prasannan, Priya and Suliman, Huda S. and Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu},
abstractNote = {Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.},
doi = {10.1016/J.BBRC.2009.03.098},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 4,
volume = 382,
place = {United States},
year = {2009},
month = {5}
}