title: Structural, kinetic and chemical mechanism of isocitrate dehydrogenase-1 from Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli, flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently, there is no mechanistic or structural information about ICDH. We optimized expression and purification to a yield sufficiently high to perform the first detailed kinetic and structural studies of Mtb ICDH-1. A large solvent kinetic isotope effect [(D2O)V = 3.0 ± 0.2, and (D2O)(V/Kisocitrate) = 1.5 ± 0.3] and a smaller primary kinetic isotope effect [(D)V = 1.3 ± 0.1, and (D)(V/K[2R-(2)H]isocitrate) = 1.5 ± 0.2] allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn(2+) bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking β sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyzes the formation of α-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel.
authors: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Christine E
authors: CE
authors: Quartararo
authors: Saugata
authors: S
authors: Hazra
authors: Timin
authors: T
authors: Hadi
authors: John S
authors: JS
authors: Blanchard
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D020836:Protein Structure, Quaternary; D055672:Static Electricity; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D007521:Isocitrate Dehydrogenase