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Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
16844 42617 2015 9 PDF Available
Title
Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis
Abstract

•Role of critical residues in acetohydroxyacid synthase (AHAS) was evaluated.•These conserved residues are located in the herbicide binding site of AHAS.•Kinetic result of the variants suggested that R318 play a key role in catalytic activity.•A146 and Q148 might be involved in herbicide binding of AHAS.•M512 and V513 constitute part of the herbicide-binding site.

Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold.

Keywords
TB, tuberculosis; M. tuberculosis, Mycobacterium tuberculosis; AHAS, acetohydroxyacid synthase; BCAA, branched-chain amino acid; FAD, flavin adenine dinucleotide; ThDP, thiamin diphosphate; DMSO, dimethyl sulfoxideAcetohydroxyacid Synthase; Site-Directed
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Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 78, October 2015, Pages 18–26
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