{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T13:38:05Z","timestamp":1790861885363,"version":"4.1.0"},"reference-count":30,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2019,10,28]],"date-time":"2019-10-28T00:00:00Z","timestamp":1572220800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/M017702\/1"],"award-info":[{"award-number":["BB\/M017702\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/R505894\/1"],"award-info":[{"award-number":["BB\/R505894\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The FEBS Journal"],"published-print":{"date-parts":[[2020,4]]},"abstract":"<jats:sec><jats:label\/><jats:p>In the native pathway to therapeutic cannabinoid biosynthesis in <jats:italic>Cannabis\u00a0sativa<\/jats:italic>, the three\u2010step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2\u00a0\u2192\u00a0C7 aldol condensation). In the absence of OAC, a nonenzymatic C2\u00a0\u2192\u00a0C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an \u2018aldol switch\u2019 is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable \u2018aldol switch\u2019 consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue\u2010specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high\u2010resolution structural analysis of multiple enzyme homologues with classical site\u2010directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes.<\/jats:p><\/jats:sec><jats:sec><jats:title>Enzymes<\/jats:title><jats:p>TKS, <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/EC2\/3\/1\/206.html\">EC 2.3.1.206<\/jats:ext-link>; OAC, <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/EC4\/4\/1\/26.html\">EC 4.4.1.26<\/jats:ext-link>; chalcone synthase, <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/EC2\/3\/1\/74.html\">EC 2.3.1.74<\/jats:ext-link>; stilbene synthase, <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/EC2\/3\/1\/95.html\">EC 2.3.1.95<\/jats:ext-link>; 2\u2010PS, <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/EC2\/3\/1\/-.html\">EC 2.3.1.-<\/jats:ext-link>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Accession numbers<\/jats:title><jats:p>The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.rcsb.org\/pdb\/search\/structidSearch.do?structureId=6GW3\">6GW3<\/jats:ext-link>.<\/jats:p><\/jats:sec>","DOI":"10.1111\/febs.15089","type":"journal-article","created":{"date-parts":[[2019,10,12]],"date-time":"2019-10-12T19:32:57Z","timestamp":1570908777000},"page":"1511-1524","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":36,"title":["Structure of the <i>Cannabis\u00a0sativa<\/i> olivetol\u2010producing enzyme reveals cyclization plasticity in type III polyketide synthases"],"prefix":"10.1111","volume":"287","author":[{"given":"Lewis J.","family":"Kearsey","sequence":"first","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole","family":"Prandi","sequence":"additional","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vijaykumar","family":"Karuppiah","sequence":"additional","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cunyu","family":"Yan","sequence":"additional","affiliation":[{"name":"BBSRC\/EPSRC Synthetic Biology Research Centre SYNBIOCHEM The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Leys","sequence":"additional","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helen","family":"Toogood","sequence":"additional","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eriko","family":"Takano","sequence":"additional","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"},{"name":"BBSRC\/EPSRC Synthetic Biology Research Centre SYNBIOCHEM The University of Manchester UK"},{"name":"EPSRC\/BBSRC Future Biomanufacturing Research Hub The University of Manchester UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4182-3500","authenticated-orcid":false,"given":"Nigel S.","family":"Scrutton","sequence":"additional","affiliation":[{"name":"Manchester Institute of Biotechnology and School of Chemistry The University of Manchester UK"},{"name":"BBSRC\/EPSRC Synthetic Biology Research Centre SYNBIOCHEM The University of Manchester UK"},{"name":"EPSRC\/BBSRC Future Biomanufacturing Research Hub The University of Manchester 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