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Role of a highly conserved proline-126 in ThDP binding of Mycobacterium tuberculosis acetohydroxyacid synthase

Title
Role of a highly conserved proline-126 in ThDP binding of Mycobacterium tuberculosis acetohydroxyacid synthase
Author
고성호
Keywords
Acetohydroxyacid synthase; ThDP dependent enzyme; ThDP binding; Mutagenesis; Homology modeling
Issue Date
2013-09
Publisher
Elsevier Science B.V., Amsterdam.
Citation
Enzyme and microbial technology, 2013, 53(4), p.243-249
Abstract
Acetohydroxyacid synthase (AHAS) of Mycobacterium tuberculosis is a promising target for the development of anti-tuberculosis agents. With the absence of an available bacterial AHAS crystal structure,that of M. tuberculosis, site-directed mutagenesis has been a useful tool for determining its structural and functional features. In this study, a highly conserved proline residue (P126 of M. tuberculosis AHAS) was selected, and the possible role was evaluated by site-directed mutagenesis. P126 was replaced by valine, threonine, alanine, and glutamate to yield P126V, P126T, P126A, and P126E, respectively. All variants were expressed in their soluble forms in Escherichia coli and purified to near homogeneity. The molecular mass (SDS-PAGE) of the purified variants was ∼68 kDa, which is similar to that of wild-type AHAS. The P126V, P126T, and P126A variants exhibited significantly lower activity than wild-type AHAS,whereas P126E was inactive under the tested assay conditions. Furthermore, the P126V and P126T variants showed a significantly decreased preference toward pyruvate and ThDP as substrate and cofactor respectively, whereas the P126A showed similar kinetics to that of wild-type AHAS. Like in AHAS from yeast Saccharomyces cerevisiae (PDB ID: 1N0H), residue P126 is located in the ThDP binding pocket of M. tuberculosis AHAS homology model. Collectively, these results suggest that the conserved P126 plays asignificant role in the ThDP binding of M. tuberculosis AHAS.
URI
https://www.sciencedirect.com/science/article/pii/S0141022913001270?via%3Dihubhttp://hdl.handle.net/20.500.11754/50457
ISSN
0141-0229
DOI
10.1016/j.enzmictec.2013.05.006
Appears in Collections:
COLLEGE OF MEDICINE[S](의과대학) > ETC
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