Role of a highly conserved proline-126 in ThDP binding of Mycobacterium tuberculosis acetohydroxyacid synthase.
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 a significant role in the ThDP binding of M. tuberculosis AHAS.
Department of Chemistry, Hanyang University, Seoul, South Korea.
Irshad Ahmed
IA
Baig
Vinayakumar
V
Gedi
Sang-Choon
SC
Lee
Seong-Ho
SH
Koh
Moon-Young
MY
Yoon
D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
D001426:Bacterial Proteins; D011392:Proline; D000095:Acetolactate Synthase; D013835:Thiamine Pyrophosphate
Acetohydroxyacid synthase; Homology modeling; Mutagenesis; ThDP binding; ThDP dependent enzyme