Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.
Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.
Department of Chemistry, Hanyang University, Seoul, South Korea.
Won-Mook
WM
Lim
Irshad Jameel
IJ
Baig
Im Joung
IJ
La
Jung-Do
JD
Choi
Dong-Eun
DE
Kim
Sung-Kun
SK
Kim
Jae-Wook
JW
Hyun
Giyoung
G
Kim
Chang-Ho
CH
Kang
Young Jin
YJ
Kim
Moon-Young
MY
Yoon
D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000493:pharmacokinetics; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D012764:Shigella sonnei / Q000201:enzymology* / Q000235:genetics
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D008024:Ligands; D000095:Acetolactate Synthase