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Site-directed mutagenesis of catalytic and regulatory subunits of Mycobacterium tuberculosis acetohydroxyacid synthase

Paper ID Volume ID Publish Year Pages File Format Full-Text
17646 42686 2010 5 PDF Available
Title
Site-directed mutagenesis of catalytic and regulatory subunits of Mycobacterium tuberculosis acetohydroxyacid synthase
Abstract

The acetohydroxyacid synthase (AHAS), which is involved in the biosynthesis of branched-chain amino acids (BCAAs), is the target of several classes of herbicides. The catalytic (CSU) and regulatory subunits (RSU) of Mycobacterium tuberculosis AHAS (MtbAHAS) were cloned, expressed, and purified to homogeneity. A homology model of MtbAHAS CSU showed three residues (L141, F147 and W516) at the sulfonylurea (SU) herbicide binding site. The residues were mutated and the variant enzymes characterized with respect to its catalytic properties and sensitivity to two SU herbicides. All the tested mutants showed a decrease in Vmax compared to the wild-type protein. The mutants (F147A, F147R, and W516R) showed strong resistance to the two SU herbicides tested, indicating that the compounds related to these herbicides which target these critical residues, may serve as potent and specific anti-tuberculosis drugs. Furthermore, among the mutants of RSU (S27A, L89A and R101A), the S27A mutation caused 56-fold decrease in Vmax of the holoenzyme, whereas the L89A and R101A showed 4- and 12-fold decrease, respectively. The holoenzymes with S27A and L89A showed resistance to leucine. These results reveal characteristics of SU herbicide-resistant mutants of the CSU, and catalytically important residues of the RSU in MtbAHAS.

Keywords
AHAS, acetohydroxyacid synthase; BCAAs, branched-chain amino acids; CSU, catalytic subunit; RSU, regulatory subunit; MtbAHAS, Mycobacterium tuberculosis AHAS; wtCSU, wild-type catalytic subunit; SU, sulfonylurea; IPTG, isopropyl-β-d-thiogalactosideAcetohy
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Site-directed mutagenesis of catalytic and regulatory subunits of Mycobacterium tuberculosis acetohydroxyacid synthase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 46, Issues 3–4, 5 March 2010, Pages 304–308
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us