Toxoplasmosis is a widespread parasitic disease caused by Toxoplasma gondii, an obligate intracellular protozoa belonging to the phylum Apicomplexa. Toxoplasmosis is a major public health problem, infecting one-third of humans worldwide. Due to the fact that no effective vaccine is currently available and treatment is based on drugs for which resistance is emerging, there is an urgent need to discover novel drug targets that are exploitable for the design of new therapeutics against the pathogen. A recent proteomic analysis of partially sporulated oocysts of T. gondii showed that oocysts have a greater capability of de novo amino acid biosynthesis, shedding light on several stage-specific proteins whose functional profile is in accord with the oocyst need to resist various environmental stresses [1]. Herein, we focused our attention on two enzymes belonging to these putative oocyst/sporozoite-specific protein group: the ornithine aminotransferase (OAT) and the cystathionine γ-lyase (CGL). OAT is involved in the polyamine metabolism and catalyzes the reversible conversion of L-ornithine into glutamate-5-semialdehyde and glutamate, while CGL catalyzes the cleavage of L-cystathionine (L-cth) to L-cysteine, α-ketobutyrate and ammonia in the reverse transsulfuration pathway. Despite the central metabolic roles of these enzymes, the functionality of none of them has so far been investigated. Herein, a biochemical characterization of OAT and CGL from T. gondii has been performed, in order to expand the very limited knowledge about the polyamine and cysteine metabolism of the parasite and to explore the possible use of these enzymes as novel drug targets against toxoplasmosis. Analysis of spectral and kinetic properties of TgOAT revealed that the enzyme is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not possess a specific ornithine aminotransferase activity, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity, highlighting its possible role both in 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 resulted in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The purified TgCGL is a functional enzyme which splits L-cth almost exclusively at the CγS bond to yield L-cysteine. This finding likely implies that the reverse transsulfuration pathway is operative in the parasite. The enzyme displays only marginal reactivity toward L-cysteine, which is also a mixed-type inhibitor of TgCGL activity, therefore indicating a tight regulation of cysteine intracellular levels in the parasite. Structure-guided homology modelling revealed two striking amino acid differences between human and TgCGL active sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of these two residues to the corresponding residues in human revealed their importance in modulating both substrate and reaction specificity of the parasitic enzyme. Altogether our findings could be considered as a first step toward exploring the possible use of TgOAT and TgCGL as an anti-toxoplasmosis drug targets.

Biochemical characterization of ornithine aminotransferase and cystathionine γ-lyase from Toxoplasma gondii: possible targets for drug development?

Maresi, Elena
2018

Abstract

Toxoplasmosis is a widespread parasitic disease caused by Toxoplasma gondii, an obligate intracellular protozoa belonging to the phylum Apicomplexa. Toxoplasmosis is a major public health problem, infecting one-third of humans worldwide. Due to the fact that no effective vaccine is currently available and treatment is based on drugs for which resistance is emerging, there is an urgent need to discover novel drug targets that are exploitable for the design of new therapeutics against the pathogen. A recent proteomic analysis of partially sporulated oocysts of T. gondii showed that oocysts have a greater capability of de novo amino acid biosynthesis, shedding light on several stage-specific proteins whose functional profile is in accord with the oocyst need to resist various environmental stresses [1]. Herein, we focused our attention on two enzymes belonging to these putative oocyst/sporozoite-specific protein group: the ornithine aminotransferase (OAT) and the cystathionine γ-lyase (CGL). OAT is involved in the polyamine metabolism and catalyzes the reversible conversion of L-ornithine into glutamate-5-semialdehyde and glutamate, while CGL catalyzes the cleavage of L-cystathionine (L-cth) to L-cysteine, α-ketobutyrate and ammonia in the reverse transsulfuration pathway. Despite the central metabolic roles of these enzymes, the functionality of none of them has so far been investigated. Herein, a biochemical characterization of OAT and CGL from T. gondii has been performed, in order to expand the very limited knowledge about the polyamine and cysteine metabolism of the parasite and to explore the possible use of these enzymes as novel drug targets against toxoplasmosis. Analysis of spectral and kinetic properties of TgOAT revealed that the enzyme is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not possess a specific ornithine aminotransferase activity, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity, highlighting its possible role both in 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 resulted in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The purified TgCGL is a functional enzyme which splits L-cth almost exclusively at the CγS bond to yield L-cysteine. This finding likely implies that the reverse transsulfuration pathway is operative in the parasite. The enzyme displays only marginal reactivity toward L-cysteine, which is also a mixed-type inhibitor of TgCGL activity, therefore indicating a tight regulation of cysteine intracellular levels in the parasite. Structure-guided homology modelling revealed two striking amino acid differences between human and TgCGL active sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of these two residues to the corresponding residues in human revealed their importance in modulating both substrate and reaction specificity of the parasitic enzyme. Altogether our findings could be considered as a first step toward exploring the possible use of TgOAT and TgCGL as an anti-toxoplasmosis drug targets.
2018
Inglese
93
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14242/113820
Il codice NBN di questa tesi è URN:NBN:IT:UNIVR-113820