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Instruments / Technical Area	Publication Details	Published	Actions
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis of the inhibition of cystathionine γ‐lyase from Toxoplasma gondii by propargylglycine and cysteine Carmen Fernández-Rodríguez , Carolina Conter , Iker Oyenarte , Filippo Favretto , Iban Quintana , Maria Luz Martinez-Chantar , Alessandra Astegno , Luis Alfonso Martinez-Cruz Protein Science DOI: 10.1002/pro.4619 Diamond Proposal Number(s): [28360] Open Access Show Abstract ▼ Abstract: Cystathionine γ-lyase (CGL) is a PLP-dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL-catalyzed process consists of an α,γ-elimination reaction that breaks down cystathionine into cysteine, α-ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2S). Importantly, inhibition of the enzyme and consequently of its H2S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as Toxoplasma gondii, the causative agent of toxoplasmosis, encode a CGL enzyme (TgCGL) that almost exclusively catalyzes the canonical process, with only minor reactivity to cysteine. Interestingly, the substitution of N360 by a serine (the equivalent amino acid residue in the human enzyme) at the active site changes the specificity of TgCGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the CγS and the CβS bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme-substrate specificity, we have elucidated the crystal structures of native TgCGL and the variant TgCGL-N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor D,L-propargylglycine (PPG). Our structures reveal the binding mode of each molecule within the catalytic cavity and help explain the inhibitory behavior of cysteine and PPG. A specific inhibitory mechanism of TgCGL by PPG is proposed.	Mar 2023
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural insight into the unique conformation of cystathionine β-synthase from Toxoplasma gondii Carmen Fernandez-Rodriguez , Iker Oyenarte , Carolina Conter , Irene Gonzalez-Recio , Reyes Núñez-Franco , Claudia Gil-Pitarch , Iban Quintana , Gonzalo Jiménez-Osés , Paola Dominici , Maria Luz Martinez-Chantar , Alessandra Astegno , Luis Alfonso Martinez-Cruz Computational And Structural Biotechnology Journal 19, 3542 - 3555 DOI: 10.1016/j.csbj.2021.05.052 Diamond Proposal Number(s): [28360] Open Access Show Abstract ▼ Abstract: Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.	Aug 2021

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