Choi, Kyoung-Jae Noh, Kyoung Mi Kim, Dong-Eun Ha, Byung Hak Kim, Eunice Eunkyung Yoon, Moon-Young 2024-01-21T00:30:54Z 2024-01-21T00:30:54Z 2021-08-31 2007-10-01 0003-9861 https://pubs.kist.re.kr/handle/201004/134052 Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate- (ThDP)- and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids (BCAAs) leucine, isoleucine, and valine. The gene from Haemophilus influenzae that encodes the AHAS catalytic subunit was cloned, overexpressed in Escherichia coli BL21(DE3), and purified to homogeneity. The purified H. influenzae AHAS catalytic subunit (Hin-AHAS) appeared as a single band on SIDS-PAGE gel, with a molecular mass of approximately 63 kDa. The enzyme catalyzes the condensation of two molecules of pyruvate to form acetolactate, with a K,, of 9.2 mM and the specific activity of 1.5 mu mol/min/mg. The cofactor activation constant (K-c = 13.5 mu M) and the dissociation constant (K-d = 3.3 mu M) of ThDP were also determined by enzymatic assay and tryptophan fluorescence quenching studies, respectively. We screened a chemical library to discover new inhibitors of the Hin AHAS catalytic subunit. Through which, AVS-2087 (IC50 = 0.53 mu M), KSW30191 IC50 = 1.42 mu M), and KHG20612 (IC50 = 4.91 mu M) displayed potent inhibition as compare to sulfometuron methyl (IC50 = 276.31 mu M). (c) 2007 Elsevier Inc. All rights reserved. English ELSEVIER SCIENCE INC ACID SYNTHASE ACETOLACTATE SYNTHASE ESCHERICHIA-COLI ISOZYME-III BINDING RECONSTITUTION TUBERCULOSIS DIPHOSPHATE MECHANISM ENZYME Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors Article 10.1016/j.abb.2007.07.011 1 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, v.466, no.1, pp.24 - 30 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 466 1 24 30 scie scopus 000249834100004 2-s2.0-34548639649 Biochemistry & Molecular Biology Biophysics Biochemistry & Molecular Biology Biophysics Article ACID SYNTHASE ACETOLACTATE SYNTHASE ESCHERICHIA-COLI ISOZYME-III BINDING RECONSTITUTION TUBERCULOSIS DIPHOSPHATE MECHANISM ENZYME Haemophilus influenzae acetohydroxyacid synthase catalytic subunit purification characterization inhibitor screening