Molecular basis of the substrate specificity and the catalytic mechanism of citramalate synthase from Leptospira interrogans.
Leptospira interrogans is the causative agent for leptospirosis, a zoonotic disease of global importance. In contrast with most other micro-organisms, L. interrogans employs a pyruvate pathway to synthesize isoleucine and LiCMS (L. interrogans citramalate synthase) catalyses the first reaction of the pathway which converts pyruvate and acetyl-CoA into citramalate, thus making it an attractive target for the development of antibacterial agents. We report here the crystal structures of the catalytic domain of LiCMS and its complexes with substrates, and kinetic and mutagenesis studies of LiCMS, which together reveal the molecular basis of the high substrate specificity and the catalytic mechanism of LiCMS. The catalytic domain consists of a TIM barrel flanked by an extended C-terminal region. It forms a homodimer in the crystal structure, and the active site is located at the centre of the TIM barrel near the C-terminal ends of the beta-strands and is composed of conserved residues of the beta-strands of one subunit and the C-terminal region of the other. The substrate specificity of LiCMS towards pyruvate against other alpha-oxo acids is dictated primarily by residues Leu(81), Leu(104) and Tyr(144), which form a hydrophobic pocket to accommodate the C(2)-methyl group of pyruvate. The catalysis follows the typical aldol condensation reaction, in which Glu(146) functions as a catalytic base to activate the methyl group of acetyl-CoA to form an enolated acetyl-CoA intermediate and Arg(16) as a general acid to stabilize the intermediate.
Ma
Jun
J
Laboratory of Microbial Molecular Physiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng-Lin Road, Shanghai 200032, China.
Zhang
Peng
P
Zhang
Zilong
Z
Zha
Manwu
M
Xu
Hai
H
Zhao
Guoping
G
Ding
Jianping
J
D000123:Acetyltransferases; D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D003460:Crystallization; D007700:Kinetics; D007921:Leptospira interrogans / Q000201:enzymology*; D008314:Malonates / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D008314:Malonates; C030290:malonic acid; D000217:Acyltransferases; D000123:Acetyltransferases; C116922:(R)-citramalate synthase