Cargando…

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 β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways f...

Descripción completa

Detalles Bibliográficos
Autores principales: Maresi, Elena, Janson, Giacomo, Fruncillo, Silvia, Paiardini, Alessandro, Vallone, Rosario, Dominici, Paola, Astegno, Alessandra
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073527/
https://www.ncbi.nlm.nih.gov/pubmed/30036991
http://dx.doi.org/10.3390/ijms19072111
_version_ 1783344211172524032
author Maresi, Elena
Janson, Giacomo
Fruncillo, Silvia
Paiardini, Alessandro
Vallone, Rosario
Dominici, Paola
Astegno, Alessandra
author_facet Maresi, Elena
Janson, Giacomo
Fruncillo, Silvia
Paiardini, Alessandro
Vallone, Rosario
Dominici, Paola
Astegno, Alessandra
author_sort Maresi, Elena
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6073527
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-60735272018-08-13 Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii Maresi, Elena Janson, Giacomo Fruncillo, Silvia Paiardini, Alessandro Vallone, Rosario Dominici, Paola Astegno, Alessandra Int J Mol Sci Article 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. MDPI 2018-07-20 /pmc/articles/PMC6073527/ /pubmed/30036991 http://dx.doi.org/10.3390/ijms19072111 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Maresi, Elena
Janson, Giacomo
Fruncillo, Silvia
Paiardini, Alessandro
Vallone, Rosario
Dominici, Paola
Astegno, Alessandra
Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
title Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
title_full Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
title_fullStr Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
title_full_unstemmed Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
title_short Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
title_sort functional characterization and structure-guided mutational analysis of the transsulfuration enzyme cystathionine γ-lyase from toxoplasma gondii
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073527/
https://www.ncbi.nlm.nih.gov/pubmed/30036991
http://dx.doi.org/10.3390/ijms19072111
work_keys_str_mv AT maresielena functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii
AT jansongiacomo functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii
AT fruncillosilvia functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii
AT paiardinialessandro functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii
AT vallonerosario functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii
AT dominicipaola functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii
AT astegnoalessandra functionalcharacterizationandstructureguidedmutationalanalysisofthetranssulfurationenzymecystathionineglyasefromtoxoplasmagondii