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

Abstract:

: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.

journal_name

Enzyme Microb Technol

authors

Jung IP,Cho JH,Koo BS,Yoon MY

doi

10.1016/j.enzmictec.2015.06.009

subject

Has Abstract

pub_date

2015-10-01 00:00:00

pages

18-26

eissn

0141-0229

issn

1879-0909

pii

S0141-0229(15)30014-4

journal_volume

78

pub_type

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