Research paper Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase June-Haeng Cho 1 , Mi-Young Lee 1 , Irshad Ahmed Baig, Na-Reum Ha, Joungmok Kim 2 , Moon-Young Yoon * Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea article info Article history: Received 8 October 2012 Accepted 10 March 2013 Available online 22 March 2013 Keywords: AHAS Inhibitors Pseudomonas aeruginosa MIC BCAAs abstract Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohy- droxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and charac- terized the catalytic subunit (CSU) 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 on SDS-PAGE. Enzyme kinetics showed that the enzyme has a K m of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37  C and a pH of 7.5. The K d for thiamine diphosphate (ThDP) was 89.92  17.9 mM, as determined by fluorescence quenching. The cofactor activation constants (K s ) for ThDP and (K c ) for Mg 2þ were 0.6  0.1 and 560.8  7.4 mM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These com- pounds inhibit nearly 100% of AHAS activity, with IC 50 values of 1.19 mM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 mg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873  A. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial thera- peutics. Results of the current study provide an impetus to further evaluate the potency of these in- hibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Pseudomonas aeruginosa (P. aeruginosa) is a versatile Gram- negative bacterium that lives in soil, marshes, and coastal marine habitats, as well as on plant and animal tissue [1,2]. The emergence of P. aeruginosa as a major opportunistic human pathogen may be a consequence of its resistance to antibiotics and disinfectants that eliminate other environmental bacteria [3]. P. aeruginosa is a cause of bacteremia in burns, urinary-tract infections, and hospital-acquired pneumonia in patients on respirators [4]. It is also the predomi- nant cause of morbidity and mortality in cystic fibrosis patients, whose abnormal airway epithelia allow P. aeruginosa to establish long-term colonies in the lungs [5]. These infections are impossible to eradicate, in part because the bacterium is naturally resistant to antibiotics. Ultimately, these infections lead to pulmonary failure and death. Therefore, there is an immense need to develop an effective antibiotic against drug resistant strains of P. aeruginosa. Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase) catalyzes the first step in the biosynthesis of Abbreviations: AHAS, acetohydroxyacid synthase; BCAAs, branched-chain amino acids; CSU, catalytic subunit; P. aeruginosa, Pseudomonas aeruginosa; MIC, minimum inhibitory concentration; ThDP, thiamine diphosphate. * Corresponding author. Department of Chemistry, Hanyang University, Hang- dang dong, Sung-dong gu, Seoul 133-791, Republic of Korea. Tel.: þ82 2 2220 0946; fax: þ82 2 2299 0763. E-mail address: myyoon@hanyang.ac.kr (M.-Y. Yoon). 1 Both the authors contributed equally. 2 Present address: Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea. Contents lists available at SciVerse ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.03.007 Biochimie 95 (2013) 1411e1421