Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans.
LiCMS (Leptospira interrogans citramalate synthase) catalyses the first reaction of the isoleucine biosynthesis pathway in L. interrogans, the pathogen of leptospirosis. The catalytic reaction is regulated through feedback inhibition by its end product isoleucine. To understand the molecular basis of the high selectivity of the inhibitor and the mechanism of feedback inhibition, we determined the crystal structure of LiCMSC (C-terminal regulatory domain of LiCMS) in complex with isoleucine, and performed a biochemical study of the inhibition of LiCMS using mutagenesis and kinetic methods. LiCMSC forms a dimer of dimers in both the crystal structure and solution and the dimeric LiCMSC is the basic functional unit. LiCMSC consists of six beta-strands forming two anti-parallel beta-sheets and two alpha-helices and assumes a betaalphabeta three-layer sandwich structure. The inhibitor isoleucine is bound in a pocket at the dimer interface and has both hydrophobic and hydrogen-bonding interactions with several conserved residues of both subunits. The high selectivity of LiCMS for isoleucine over leucine is primarily dictated by the residues, Tyr430, Leu451, Tyr454, Ile458 and Val468, that form a hydrophobic pocket to accommodate the side chain of the inhibitor. The binding of isoleucine has inhibitory effects on the binding of both the substrate, pyruvate, and coenzyme, acetyl-CoA, in a typical pattern of K-type inhibition. The structural and biochemical data from the present study together suggest that the binding of isoleucine affects the binding of the substrate and coenzyme at the active site, possibly via conformational change of the dimer interface of the regulatory domain, leading to inhibition of the catalytic reaction.
State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Peng
P
Zhang
Jun
J
Ma
Zilong
Z
Zhang
Manwu
M
Zha
Hai
H
Xu
Guoping
G
Zhao
Jianping
J
Ding
D000123:Acetyltransferases; D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D025461:Feedback, Physiological; D015971:Gene Expression Regulation, Enzymologic; D007532:Isoleucine; D007921:Leptospira interrogans / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation
D007532:Isoleucine; D000217:Acyltransferases; D000123:Acetyltransferases; C116922:(R)-citramalate synthase