Pseudomonas aeruginosa의 Acetohydroxyacid synthase 촉매인자에 대한 특성 및 저해 연구

Abstract

Microbes and plants synthesize essential branched chain amino acids such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). AHAS was recently identified as a potential anti-bacterial target. To help find an effective AHAS inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit of Pseudomonas aeruginosa AHAS and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme as determined by SDS-PAGE. Enzyme kinetics revealed a Km of 14.2 mM and a specific activity of 0.12 U/mg. The optimum temperature for activity was 37oC, and the optimum pH was 7.5. The Kd when combined with thiamine diphosphate (ThDP) was 89.92 ± 17.9 mM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg2+ were 0.6 ± 0.1 and 560.8 ± 7.4 mM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236 and AVS2397 are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of the AHAS activity, with IC50 values of 1.19 µM, 5.0 nM, 25 nM, and 13 nM respectively. Furthermore, one of the potent compounds, AVS 2093, showed growth inhibition with minimal inhibitory concentrations (MIC) of 742.9 µg/ml against the ATCC 9027 strain of P. aeruginosa. The correlation of the inhibitory activity of the AVS 2093 compound against P. aeruginosa AHAS with the inhibition of cell growth suggests that AHAS might be a target protein for the development of novel anti-bacterial therapeutics. The results of this study provide an impetus to further evaluate the potency of these inhibitors against pathogenic Pseudomonas aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.