TAILIEUCHUNG - Báo cáo khoa học: Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC ) is a key enzyme in the folate biosyn-thetic pathway. Information regarding key residues in the dihydrofolate-binding site ofMycobacterium aviumdihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as resi-dues that are potentially important in interactions with dihydrofolate and antifolates (. trimethoprim), respectively. | ễFEBS Journal Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase Ronnie A. Bỏck1 Jose L. Soulages2 and William W. Barrow1 1 Department of Veterinary Pathobiology Center for Veterinary Health Sciences Oklahoma State University Stillwater OK USA 2 Department of Biochemistry and Molecular Biology Noble Research Center Oklahoma State University Stillwater OK USA Keywords dihydrofolate reductase mycobacteria site-directed mutagenesis trimethoprim Correspondence W. W. Barrow Department of Veterinary Pathobiology 250 McElroy Hall Center for Veterinary Health Sciences Oklahoma State University Stillwater OK 74078 USA Fax 1 405 744 3738 Tel 1 405 744 1842 E-mail Website http Present address Department of Biology University of Namibia Windhoek Namibia Received 24 february 2007 revised 22 April 2007 accepted 30 April2007 doi Dihydrofolate reductase EC is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates . trimethoprim respectively. Asp31 and Leu32 were modified by site-directed mutagenesis giving the mutants D31A D31E D31Q D31N and D31L and L32A L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21 DE3 pLysS and purified using His-Bind resin functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function D31E D31Q and D31N reduced activity by 80-90 and D31A and D31L by 90 . All D31 mutants had modified kinetics ranging from three-fold D31N to 283-fold D31L increases in Km for dihydrofolate and 12-fold D31N to .

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