title: Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex.
abstract: The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10-80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the shikimate dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP(+)-dependent oxidation of shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the 'core' structure of the enzyme.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2.
authors.forename: James
authors.initials: J
authors.lastname: Peek
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2.
authors.forename: Gianni
authors.initials: G
authors.lastname: Castiglione
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2.
authors.forename: Thomas
authors.initials: T
authors.lastname: Shi
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2; Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario, Canada M5S 3B2. Electronic address: dinesh.christendat@utoronto.ca.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Christendat
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D014644:Genetic Variation; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012765:Shikimic Acid / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D012765:Shikimic Acid; D009249:NADP; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase
keywords: AROM complex; Enzyme inhibition; Enzyme kinetics; Shikimate dehydrogenase; Shikimate pathway; Toxoplasma gondii