{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T22:38:10Z","timestamp":1781044690582,"version":"3.54.1"},"reference-count":31,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2023,5,10]],"date-time":"2023-05-10T00:00:00Z","timestamp":1683676800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteins"],"published-print":{"date-parts":[[2023,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Cystathionine \u03b2\u2010synthase (CBS) catalyzes the condensation of <jats:sc>l<\/jats:sc>\u2010serine and <jats:sc>l<\/jats:sc>\u2010homocysteine to give <jats:sc>l<\/jats:sc>\u2010cystathionine in the transsulfuration pathway. Recently, a few <jats:italic>O<\/jats:italic>\u2010acetylserine (<jats:sc>l<\/jats:sc>\u2010OAS)\u2010dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with <jats:sc>l<\/jats:sc>\u2010OAS. CBS from <jats:italic>Toxoplasma gondii<\/jats:italic> (<jats:italic>Tg<\/jats:italic>CBS) can efficiently use both <jats:sc>l<\/jats:sc>\u2010serine and <jats:sc>l<\/jats:sc>\u2010OAS to form <jats:sc>l<\/jats:sc>\u2010cystathionine. In this work, a series of site\u2010specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of <jats:sc>l<\/jats:sc>\u2010serine\/<jats:sc>l<\/jats:sc>\u2010OAS preference in <jats:italic>Tg<\/jats:italic>CBS. We found that the S84A\/Y160F\/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing \u03b2\u2010replacement activity only with <jats:sc>l<\/jats:sc>\u2010OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with <jats:sc>l<\/jats:sc>\u2010serine is prevented by the triple mutation, indicating the importance of the H\u2010bonds between the hydroxyl groups of Y160, Y246, and S84 with <jats:sc>l<\/jats:sc>\u2010serine in formation of the intermediate. Analysis of the independent effect of each mutation on <jats:italic>Tg<\/jats:italic>CBS activity and investigation of the protein\u2013aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the <jats:sc>l<\/jats:sc>\u2010serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in <jats:italic>Tg<\/jats:italic>CBS will facilitate the design of new antimicrobial compounds.<\/jats:p>","DOI":"10.1002\/prot.26507","type":"journal-article","created":{"date-parts":[[2023,5,11]],"date-time":"2023-05-11T04:55:46Z","timestamp":1683780946000},"page":"1383-1393","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Key substrate recognition residues in the active site of cystathionine beta\u2010synthase from <i>Toxoplasma gondii<\/i>"],"prefix":"10.1002","volume":"91","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5999-8667","authenticated-orcid":false,"given":"Carolina","family":"Conter","sequence":"first","affiliation":[{"name":"Department of Biotechnology University of Verona  Verona Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Filippo","family":"Favretto","sequence":"additional","affiliation":[{"name":"Department of Biotechnology University of Verona  Verona Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paola","family":"Dominici","sequence":"additional","affiliation":[{"name":"Department of Biotechnology University of Verona  Verona Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis Alfonso","family":"Martinez\u2010Cruz","sequence":"additional","affiliation":[{"name":"Center for Cooperative Research in Biosciences (CIC bioGUNE) Basque Research and Technology Alliance (BRTA)  Derio Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7341-0970","authenticated-orcid":false,"given":"Alessandra","family":"Astegno","sequence":"additional","affiliation":[{"name":"Department of Biotechnology University of Verona  Verona 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