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The canonical CGL\u2010catalyzed process consists of an \u03b1,\u03b3\u2010elimination reaction that breaks down cystathionine into cysteine, \u03b1\u2010ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H<jats:sub>2<\/jats:sub>S). Importantly, inhibition of the enzyme and consequently of its H<jats:sub>2<\/jats:sub>S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as <jats:italic>Toxoplasma gondii<\/jats:italic>, the causative agent of toxoplasmosis, encode a CGL enzyme (<jats:italic>Tg<\/jats:italic>CGL) 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 <jats:italic>Tg<\/jats:italic>CGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the C\u03b3S and the C\u03b2S bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme\u2010substrate specificity, we have elucidated the crystal structures of native <jats:italic>Tg<\/jats:italic>CGL and the variant <jats:italic>Tg<\/jats:italic>CGL\u2010N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor <jats:sc>d<\/jats:sc>,<jats:sc>l<\/jats:sc>\u2010propargylglycine (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 <jats:italic>Tg<\/jats:italic>CGL by PPG is proposed.<\/jats:p>","DOI":"10.1002\/pro.4619","type":"journal-article","created":{"date-parts":[[2023,3,8]],"date-time":"2023-03-08T08:47:51Z","timestamp":1678265271000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Structural basis of the inhibition of cystathionine \u03b3\u2010lyase from <i>Toxoplasma gondii<\/i> by propargylglycine and cysteine"],"prefix":"10.1002","volume":"32","author":[{"given":"Carmen","family":"Fern\u00e1ndez\u2010Rodr\u00edguez","sequence":"first","affiliation":[{"name":"Center for Cooperative Research in Biosciences (CIC bioGUNE) Basque Research and Technology Alliance (BRTA)  Derio 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