Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii.

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine ß-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,ß- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes.

Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii.

Toxoplasma gondii is 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 from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and 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 activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo The 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.

Structural plasticity of calmodulin on the surface of CaF2 nanoparticles preserves its biological function.

Nanoparticles are increasingly used in biomedical applications and are especially attractive as biocompatible and biodegradable protein delivery systems. Herein, the interaction between biocompatible 25 nm CaF2 nanoparticles and the ubiquitous calcium sensor calmodulin has been investigated in order to assess the potential of these particles to serve as suitable surface protein carriers. Calmodulin is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells by changing its conformation in a calcium-dependent manner. Isothermal titration calorimetry and circular dichroism studies have shown that the interaction between calmodulin and CaF2 nanoparticles occurs with physiologically relevant affinity and that the binding process is fully reversible, occurring without significant alterations in protein secondary and tertiary structures. Experiments performed with a mutant form of calmodulin having an impaired Ca(2+)-binding ability in the C-terminal lobe suggest that the EF-hand Ca(2+)-binding motifs are directly involved in the binding of calmodulin to the CaF2 matrix. The residual capability of nanoparticle-bound calmodulin to function as a calcium sensor protein, binding to and altering the activity of a target protein, was successfully probed by biochemical assays. Even if efficiently carried by CaF2 nanoparticles, calmodulin may dissociate, thus retaining the ability to bind the peptide encompassing the putative C-terminal calmodulin-binding domain of glutamate decarboxylase and activate the enzyme. We conclude that the high flexibility and structural plasticity of calmodulin are responsible for the preservation of its function when bound in high amounts to a nanoparticle surface.