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The active ingredient for psychotropic effects in this plant is salvinorin A, a potent and highly selective \u03ba\u2010opioid receptor agonist. Salvinorin A is distinct from other well\u2010known opioids, such as morphine and codeine, in that it is a non\u2010nitrogenous diterpenoid with no affinity for \u03bc\u2010opioid receptor, the prime receptor of alkaloidal opioids. A terpene opioid that selectively targets a new opioid receptor (\u03ba\u2010opioid receptor) can be instrumental in developing alternative analgesics. Elucidation of the salvinorin A biosynthetic pathway can help bio\u2010manufacture diverse semi\u2010synthetic derivatives of salvinorin A but, to date, only two enzymes in the Salvinorin A pathway have been identified. Here, we identify CYP728D26 that catalyzes a C18 oxygenation on crotonolide G, which bears a clerodane backbone. Biochemical identity of CYP728D26 was validated by in vivo reconstitution in yeast, <jats:sup>1<\/jats:sup>H\u2010 and <jats:sup>13<\/jats:sup>C\u2010NMR analyses of the purified product, and kinetic analysis of CYP728D26 with a <jats:italic>K<\/jats:italic><jats:sub><jats:italic>m<\/jats:italic><\/jats:sub> value of 13.9\u2009\u03bcM. Beyond the single oxygenation on C18, collision\u2010induced dissociation analysis suggested two additional oxygenations are catalyzed by CYP728D26 to form crotonoldie G acid, although this carboxylic acid form is a minor product. Its close homologue CYP728D25 exhibited a C1\u2010hydroxylation on the clerodane backbone in a reconstituted yeast system. However, CYP728D25 showed no activity in in vitro assays. This result implies that catalytic activities observed from overexpression systems should be interpreted cautiously. This work identified a new CYP catalyst and advanced our knowledge of salvinorin A biosynthesis.<\/jats:p>","DOI":"10.1111\/ppl.14569","type":"journal-article","created":{"date-parts":[[2024,10,8]],"date-time":"2024-10-08T09:12:53Z","timestamp":1728378773000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Identification of clerodane diterpene modifying cytochrome <scp>P450<\/scp> (<scp>CYP728D26<\/scp>) in <i>Salvia divinorum<\/i> \u2010 en route to psychotropic salvinorin A biosynthesis"],"prefix":"10.1111","volume":"176","author":[{"given":"Iris","family":"Ngo","sequence":"first","affiliation":[{"name":"Department of Biological Sciences University of Calgary, 2500 University Dr. NW  Calgary Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rahul","family":"Kumar","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Calgary, 2500 University Dr. NW  Calgary Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liang","family":"Li","sequence":"additional","affiliation":[{"name":"The Metabolomics Innovation Centre and Department of Chemistry University of Alberta  Edmonton Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Seon\u2010Won","family":"Kim","sequence":"additional","affiliation":[{"name":"Division of Applied Life Science (BK21 Four) ABC\u2010RLRC, RIMA, Gyeongsang National University  Jinju Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0862-2149","authenticated-orcid":false,"given":"Moonhyuk","family":"Kwon","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Calgary, 2500 University Dr. NW  Calgary Alberta Canada"},{"name":"Division of Applied Life Science (BK21 Four) ABC\u2010RLRC, RIMA, Gyeongsang National University  Jinju Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1288-5347","authenticated-orcid":false,"given":"Dae\u2010Kyun","family":"Ro","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Calgary, 2500 University Dr. NW  Calgary Alberta Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,10,8]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2021.01.011"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules28093790"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1124\/jpet.107.129023"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/tpj.15304"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/jo802623n"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jep.2013.11.032"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1093\/jxb\/erw493"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(02)50957-5"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-313X.2002.01266.x"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1318"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/ol0510522"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1093\/pcp\/pcaa092"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.163.10.5617"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuropharm.2009.06.044"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.216804"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.adj3484"},{"key":"e_1_2_9_18_1","volume-title":"Some notes on the Mazatec","author":"Johnson J. 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