title: Crystal structures of 2-methylisocitrate lyase in complex with product and with isocitrate inhibitor provide insight into lyase substrate specificity, catalysis and evolution.
abstract: Two crystal structures of the C123S mutant of 2-methylisocitrate lyase have been determined, one with the bound reaction products, Mg(2+)-pyruvate and succinate, and the second with a bound Mg(2+)-(2R,3S)-isocitrate inhibitor. Comparison with the structure of the wild-type enzyme in the unbound state reveals that the enzyme undergoes a conformational transition that sequesters the ligand from solvent, as previously observed for two other enzyme superfamily members, isocitrate lyase and phosphoenolpyruvate mutase. The binding modes reveal the determinants of substrate specificity and stereoselectivity, and the stringent specificity is verified in solution using various potential substrates. A model of bound 2-methylisocitrate has been developed based on the experimentally determined structures. We propose a catalytic mechanism involving an alpha-carboxy-carbanion intermediate/transition state, which is consistent with previous stereochemical experiments showing inversion of configuration at the C(3) of 2-methylisocitrate. Structure-based sequence analysis and phylogenic tree construction reveal determinants of substrate specificity, highlight nodes of divergence of families, and predict enzyme families with new functions.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA.
authors.forename: Sijiu
authors.initials: S
authors.lastname: Liu
authors.forename: Zhibing
authors.initials: Z
authors.lastname: Lu
authors.forename: Yin
authors.initials: Y
authors.lastname: Han
authors.forename: Eugene
authors.initials: E
authors.lastname: Melamud
authors.forename: Debra
authors.initials: D
authors.lastname: Dunaway-Mariano
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
mesh_terms: D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D019755:Carbon-Carbon Lyases; C417277:methylisocitrate lyase; D008274:Magnesium