title: Subdomain II of α-isopropylmalate synthase is essential for activity: inferring a mechanism of feedback inhibition.
abstract: The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product L-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2), which exhibits a catalytic activity comparable with that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 resulted in a loss of about 50% of the catalytic activity; however, when the regulatory domain was deleted up to Arg-385, producing a protein that is almost equivalent to the intact LbIPMS2, about 90% of the activity was maintained. Moreover, in LbIPMS2 or LbIPMS1, further deletion of several residues from the C terminus of subdomain II significantly impaired or completely abolished the catalytic activity, respectively. These results define a complete and independently functional catalytic module of IPMS consisting of both the N-terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS revealed two different conformations of subdomain II that likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA.
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and Shanghai International Travel Healthcare Center, Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200335, China.
authors.forename: Zilong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and.
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and.
authors.forename: Han
authors.initials: H
authors.lastname: Yan
authors.affiliation: Key Laboratory of Medical Molecular Virology affiliated to the Ministries of Education and Health, Shanghai Medical College, and Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and.
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China, jpding@sibs.ac.cn.
authors.forename: Jianping
authors.initials: J
authors.lastname: Ding
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China, pengzhang01@sibs.ac.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology and Key Laboratory of Medical Molecular Virology affiliated to the Ministries of Education and Health, Shanghai Medical College, and Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China, Shanghai-Ministry of Science and Technology Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai 201203, China, Department of Microbiology and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region 999077, China, and gpzhao@sibs.ac.cn.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhao
mesh_terms: D015079:2-Isopropylmalate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D025461:Feedback, Physiological; D007700:Kinetics; D007919:Leptospira / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000105:Acetyl Coenzyme A; D015079:2-Isopropylmalate Synthase; D007930:Leucine
keywords: Allosteric Regulation; Catalytic Module; Cooperativity; Enzyme Catalysis; Enzyme Structure; Feedback Inhibition; Protein Conformation