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 β-strands forming two anti-parallel β-sheets and two α-helices and assumes a βαβ 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.
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Research Article|
June 12 2009
Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans Available to Purchase
Peng Zhang;
*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
†Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
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Jun Ma;
Jun Ma
1
†Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
‡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
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Zilong Zhang;
Zilong Zhang
†Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
‡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
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Manwu Zha;
Manwu Zha
*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
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Hai Xu;
Hai Xu
3
‡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
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Guoping Zhao;
Guoping Zhao
4
‡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
§Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai 201203, China
4Correspondence should be addressed to either Jianping Ding (email [email protected]) or Guoping Zhao (email [email protected]).
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Jianping Ding
Jianping Ding
4
*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
4Correspondence should be addressed to either Jianping Ding (email [email protected]) or Guoping Zhao (email [email protected]).
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Publisher: Portland Press Ltd
Received:
February 24 2009
Accepted:
April 07 2009
Accepted Manuscript online:
April 07 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 421 (1): 133–143.
Article history
Received:
February 24 2009
Accepted:
April 07 2009
Accepted Manuscript online:
April 07 2009
Citation
Peng Zhang, Jun Ma, Zilong Zhang, Manwu Zha, Hai Xu, Guoping Zhao, Jianping Ding; Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans. Biochem J 1 July 2009; 421 (1): 133–143. doi: https://doi.org/10.1042/BJ20090336
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