title: Unique substrate specificity of ornithine aminotransferase fromToxoplasma gondii.
abstract: Toxoplasma gondiiis a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine aminotransferase fromT. gondiiME49 (TgOAT). We overexpressed the protein inEscherichia coliand analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activityin vitro, suggesting a role in both arginine and GABA metabolismin vivoThe presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention.
authors.affiliation: Department of Biotechnology, University of Verona, Verona 37134, Italy alessandra.astegno@univr.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Astegno
authors.affiliation: Department of Biotechnology, University of Verona, Verona 37134, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Maresi
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona 37134, Italy.
authors.forename: Mariarita
authors.initials: M
authors.lastname: Bertoldi
authors.affiliation: Department of Biotechnology, University of Verona, Verona 37134, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: La Verde
authors.affiliation: Dipartimento di Biologia e Biotecnologie 'Charles Darwin', Sapienza Università di Roma, Piazzale Aldo Moro 5, Rome 00185, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.affiliation: Department of Biotechnology, University of Verona, Verona 37134, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Dominici
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009952:Ornithine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D014122:Toxoplasma / Q000737:chemistry / Q000201:enzymology*; D005680:gamma-Aminobutyric Acid / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; C021951:N(delta)-acetylornithine; D005680:gamma-Aminobutyric Acid; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase