Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex

• The shikimate dehydrogenase domain of the Toxoplasma gondii AROM complex (TgSDH) was recombinantly expressed.
• The isolated TgSDH domain catalyzed the oxidation of shikimate in vitro.
• The kinetic properties of the recombinant enzyme were determined.
• The sensitivity of TgSDH to potential inhibitors was evaluated.
• A structural model of TgSDH revealed a number of novel surface-exposed loops.

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.

The isolated C-terminal domain of the Toxoplasma gondii AROM complex possesses in vitro shikimate dehydrogenase (SDH) activity. Structural modeling suggests the protein has a number of unique surface-exposed loops.Figure optionsDownload high-quality image (121 K)Download as PowerPoint slide