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

Abstract

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine β-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,β- 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.

Cystathionine β-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii

“…Despite the major importance of redox balance in survival of parasites and the role that cysteine production has in redox homeostasis, to date knowledge about the cysteine biosynthetic pathway in T. gondii is still very limited 27 . The protozoan parasite includes both the CBS and CGL enzymes of the reverse transsulfuration pathway.…”

“…The protozoan parasite includes both the CBS and CGL enzymes of the reverse transsulfuration pathway. We have shown previously that the putative CGL gene encodes a functional enzyme in T. gondii 27 . Herein, we establish that the parasite possesses an intact reverse transsulfuration pathway by cloning and characterization of the gene encoding putative CBS.…”

“…The above data strongly indicate that TgCBS is an H 2 S-generating enzyme. Interestingly, while in human also CGL is primarily responsible for biogenesis of H 2 S via the hydrolysis of l-Cys, CGL in Toxoplasma has small reactivity toward l-Cys 27 . Thus, in the parasite, CGL is not likely a major source to generate H 2 S, while TgCBS is expected to be mainly involved in production of the gas.…”

“…The protozoan parasite T. gondii, the etiological agent of toxoplasmosis, includes enzymes of the reverse transsulfuration pathway CBS and CGL. We have recently demonstrated that T. gondii CGL is functional, being able to break down l-Cth into l-Cys 27 . On the other hand, T. gondii CBS has not been functionally characterized.…”

Structure of Cystathionine β-Synthase from Toxoplasma gondii, a key enzyme in its H2S production machinery

“…We recently demonstrated that both CBS and CGL from Toxoplasma gondii (TgCBS and TgCGL) are functional in toxoplasma (24), (40), implying that the reverse transsulfuration pathway is operative in the parasite. In particular, TgCBS can use both Ser and O-acetylserine (OAS) to produce Cth (24).…”