{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T19:46:50Z","timestamp":1791920810764,"version":"4.3.4"},"reference-count":39,"publisher":"American Chemical Society (ACS)","issue":"19","license":[{"start":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T00:00:00Z","timestamp":1789776000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T00:00:00Z","timestamp":1789776000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T00:00:00Z","timestamp":1789776000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100004000","name":"Guangzhou Municipal Science and Technology Program key projects","doi-asserted-by":"publisher","award":["2025A04J3976"],"award-info":[{"award-number":["2025A04J3976"]}],"id":[{"id":"10.13039\/501100004000","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22103098"],"award-info":[{"award-number":["22103098"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22473118"],"award-info":[{"award-number":["22473118"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32570459"],"award-info":[{"award-number":["32570459"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82430108"],"award-info":[{"award-number":["82430108"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Key project at central government level: the ability establishment of sustainable use for valuable Chinese medicine resources","award":["2060302"],"award-info":[{"award-number":["2060302"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Oxidosqualene cyclases (OSCs) convert linear 2,3-oxidosqualene into diverse polycyclic triterpenoids. The precise control over highly reactive carbocation intermediates, particularly the ultimate quenching mechanism (deprotonation vs hydroxylation), remains a fundamental challenge in mechanism-driven enzyme reshaping. Here, we investigate AaOSC-20433, a rare OSC from Artemisia argyi exhibiting absolute specificity for pure deprotonation to produce dammara-20,24-dien-3\u03b2-ol. This starkly contrasts with the homologous dammarenediol synthase from Panax ginseng (PgDDS), which favors hydroxylation to yield dammarenediol II. Integrating multiscale QM\/MM simulations and site-directed mutagenesis, we modeled the impact of crucial active site mutations on the reaction cascade. Our simulations revealed that S411 acts as a general base governing the terminal deprotonation. More importantly, we demonstrate that mechanism-guided active site substitutions can dramatically alter the chemical trajectory. Relaxing steric constraints (F728A) induces catalytic promiscuity to yield tricyclic and tetracyclic products. Furthermore, the L259Y mutation significantly alters the quenching network. It unlocks the hydroxylation pathway to form dammarenediol II and acts as an engineered base extending cyclization depth to yield the pentacyclic lupeol. Notably, combining these functional loci (L259Y+F728S) reveals notable synergistic epistasis between the steric cavity and the proton-transfer network, driving competitive hydration events to diversify the hydroxylated product profile. This study provides a comprehensive mechanistic framework for understanding how OSC active site mutations govern product specificity, offering a robust theoretical basis for the tailored, mechanism-driven functional reprogramming of triterpene synthases in synthetic biology.<\/jats:p>","DOI":"10.1021\/acs.jctc.6c01306","type":"journal-article","created":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T15:01:43Z","timestamp":1789830103000},"page":"10465-10474","source":"Crossref","is-referenced-by-count":0,"title":["Modeling the Impact\nof Active Site Mutations on the\nQuenching Fate and Cyclization Depth of a Rare Oxidosqualene Cyclase"],"prefix":"10.1021","volume":"22","author":[{"given":"Chenxu","family":"Liu","sequence":"first","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shun","family":"Liang","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Zheng","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangdong Pharmaceutical University , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaqi","family":"Jiao","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bing","family":"Li","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"JinCai","family":"Liang","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6986-9677","authenticated-orcid":true,"given":"Zhongqiu","family":"Liu","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1516-3497","authenticated-orcid":true,"given":"Lixin","family":"Duan","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1984-046X","authenticated-orcid":true,"given":"Ruibo","family":"Wu","sequence":"additional","affiliation":[{"name":"Sun Yat-sen University , , ,","place":["Guangzhou, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0953-7416","authenticated-orcid":true,"given":"Fan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Guangzhou University of Chinese Medicine , , ,","place":["Guangdong, China, 510006"]},{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs , ,","place":["Beijing, P. R. China, 100700"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,19]]},"reference":[{"issue":"6","key":"2026101310072513000_cit1","doi-asserted-by":"publisher","first-page":"1311","DOI":"10.1039\/b616857b","article-title":"Enzymatic\nsynthesis of cyclic triterpenes","volume":"24","author":"Abe","year":"2007","journal-title":"Nat. Prod.\nRep."},{"issue":"2","key":"2026101310072513000_cit2","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1080\/10409238.2021.1979458","article-title":"Diverse triterpene\nskeletons are derived from the expansion and divergent evolution of\n2,3-oxidosqualene cyclases in plants","volume":"57","author":"Wang","year":"2022","journal-title":"Crit.\nRev. Biochem. Mol. Biol."},{"issue":"8","key":"2026101310072513000_cit3","doi-asserted-by":"publisher","first-page":"1044","DOI":"10.1039\/C8NP00096D","article-title":"The protosteryl\nand dammarenyl cation\ndichotomy in polycyclic triterpene biosynthesis revisited: has this\n\u2018rule\u2019 finally been broken?","volume":"36","author":"Stephenson","year":"2019","journal-title":"Nat.\nProd. Rep."},{"issue":"17","key":"2026101310072513000_cit4","doi-asserted-by":"publisher","first-page":"11570","DOI":"10.1021\/acs.chemrev.7b00287","article-title":"Structural\nand Chemical Biology of Terpenoid Cyclases","volume":"117","author":"Christianson","year":"2017","journal-title":"Chem. Rev."},{"issue":"5333","key":"2026101310072513000_cit5","doi-asserted-by":"publisher","first-page":"1811","DOI":"10.1126\/science.277.5333.1811","article-title":"Structure and Function\nof a Squalene\nCyclase","volume":"277","author":"Wendt","year":"1997","journal-title":"Science"},{"key":"2026101310072513000_cit6","first-page":"1","article-title":"Chapter 1 Ginsenosides: Chemistry, Biosynthesis,\nAnalysis, and Potential\nHealth Effects","volume-title":"Advances in Food and Nutrition\nResearch","author":"Christensen","year":"2008"},{"issue":"12","key":"2026101310072513000_cit7","doi-asserted-by":"publisher","first-page":"4015","DOI":"10.1021\/acssynbio.4c00396","article-title":"De Novo Biosynthesis\nof a Polyene-Type Ginsenoside Precursor Dammaradienol in Saccharomyces\ncerevisiae","volume":"13","author":"Gan","year":"2024","journal-title":"ACS Synth. Biol."},{"issue":"22","key":"2026101310072513000_cit8","doi-asserted-by":"publisher","first-page":"5143","DOI":"10.1016\/j.febslet.2006.08.044","article-title":"Dammarenediol-II synthase,\nthe first dedicated enzyme for ginsenoside biosynthesis, in Panax\nginseng","volume":"580","author":"Tansakul","year":"2006","journal-title":"FEBS Lett."},{"key":"2026101310072513000_cit9","doi-asserted-by":"publisher","first-page":"1035","DOI":"10.1039\/c1np00006c","article-title":"Biosynthesis via\ncarbocations: theoretical studies on terpene formation","volume":"28","author":"Tantillo","year":"2011","journal-title":"Nat. Prod. Rep."},{"issue":"23","key":"2026101310072513000_cit10","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adh1418","article-title":"Discovering a uniform functional\ntrade-off of the CBC-type 2,3-oxidosqualene cyclases and deciphering\nits chemical logic","volume":"9","author":"Zhang","year":"2023","journal-title":"Sci. Adv."},{"issue":"12","key":"2026101310072513000_cit11","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2118709119","article-title":"A conserved mechanism\naffecting hydride shifting and deprotonation\nin the synthesis of hopane triterpenes as compositions of wax in oat","volume":"119","author":"Liang","year":"2022","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"3","key":"2026101310072513000_cit12","doi-asserted-by":"publisher","first-page":"2157","DOI":"10.1021\/acscatal.9b05221","article-title":"Biosynthetic Mechanism\nof Lanosterol: A Completed Story","volume":"10","author":"Diao","year":"2020","journal-title":"ACS Catal."},{"issue":"10","key":"2026101310072513000_cit13","doi-asserted-by":"publisher","first-page":"8867","DOI":"10.1021\/acscatal.9b02783","article-title":"Switching\non a Nontraditional Enzymatic Base - Deprotonation by Serine in the\nent-Kaurene Synthase from Bradyrhizobium japonicum","volume":"9","author":"Jia","year":"2019","journal-title":"ACS Catal."},{"issue":"9","key":"2026101310072513000_cit14","doi-asserted-by":"publisher","first-page":"3933","DOI":"10.1021\/acs.jcim.4c00297","article-title":"Adjusting\nCatalytic Activity of \u03b2-Amyrin Synthase GgBAS by Utilizing the\nPlasticity Residues of an Active Site","volume":"64","author":"Zheng","year":"2024","journal-title":"J. Chem.\nInf. Model."},{"issue":"8016","key":"2026101310072513000_cit15","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure\nprediction of\nbiomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"issue":"7873","key":"2026101310072513000_cit16","doi-asserted-by":"publisher","first-page":"590","DOI":"10.1038\/s41586-021-03828-1","article-title":"Highly accurate\nprotein structure prediction for the\nhuman proteome","volume":"596","author":"Tunyasuvunakool","year":"2021","journal-title":"Nature"},{"issue":"2","key":"2026101310072513000_cit17","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: improving\nthe speed and accuracy of\ndocking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"issue":"5","key":"2026101310072513000_cit18","doi-asserted-by":"publisher","first-page":"689","DOI":"10.1093\/bioinformatics\/btq007","article-title":"PyRosetta:\na script-based interface\nfor implementing molecular modeling algorithms using Rosetta","volume":"26","author":"Chaudhury","year":"2010","journal-title":"Bioinformatics"},{"issue":"6","key":"2026101310072513000_cit19","doi-asserted-by":"publisher","first-page":"1188","DOI":"10.1101\/gr.849004","article-title":"WebLogo: a sequence logo generator","volume":"14","author":"Crooks","year":"2004","journal-title":"Genome. Res."},{"issue":"16","key":"2026101310072513000_cit20","doi-asserted-by":"publisher","first-page":"1999","DOI":"10.1002\/jcc.10349","article-title":"A point-charge force\nfield for molecular mechanics simulations of proteins based on condensed-phase\nquantum mechanical calculations","volume":"24","author":"Duan","year":"2003","journal-title":"J. Comput.\nChem."},{"issue":"2","key":"2026101310072513000_cit21","doi-asserted-by":"publisher","first-page":"926","DOI":"10.1063\/1.445869","article-title":"Comparison\nof simple potential functions for simulating liquid water","volume":"79","author":"Jorgensen","year":"1983","journal-title":"J. Chem. Phys."},{"issue":"40","key":"2026101310072513000_cit22","doi-asserted-by":"publisher","first-page":"10269","DOI":"10.1021\/j100142a004","article-title":"A well-behaved electrostatic\npotential based method using charge restraints for deriving atomic\ncharges: the RESP model","volume":"97","author":"Bayly","year":"1993","journal-title":"J. Chem. Phys."},{"key":"2026101310072513000_cit23","volume-title":"Gaussian 09","author":"Frisch","year":"2009"},{"issue":"16","key":"2026101310072513000_cit24","doi-asserted-by":"publisher","first-page":"1668","DOI":"10.1002\/jcc.20290","article-title":"The Amber biomolecular simulation\nprograms","volume":"26","author":"Case","year":"2005","journal-title":"J. Comput. Chem."},{"issue":"25","key":"2026101310072513000_cit25","doi-asserted-by":"publisher","first-page":"2110","DOI":"10.1002\/jcc.25382","article-title":"3rd, Parallelization\nof CPPTRAJ enables large scale\nanalysis of molecular dynamics trajectory data","volume":"39","author":"Roe","year":"2018","journal-title":"J. Comput. Chem."},{"issue":"11","key":"2026101310072513000_cit26","doi-asserted-by":"publisher","first-page":"5047","DOI":"10.1021\/ct500381c","article-title":"POVME 2.0: An Enhanced\nTool for Determining Pocket Shape and Volume Characteristics","volume":"10","author":"Durrant","year":"2014","journal-title":"J. Chem. Theory. Comput."},{"issue":"16","key":"2026101310072513000_cit27","doi-asserted-by":"publisher","first-page":"9478","DOI":"10.1021\/acs.chemrev.9b00055","article-title":"End-Point\nBinding Free Energy Calculation with MM\/PBSA\nand MM\/GBSA: Strategies and Applications in Drug Design","volume":"119","author":"Wang","year":"2019","journal-title":"Chem. Rev."},{"issue":"9","key":"2026101310072513000_cit28","doi-asserted-by":"publisher","first-page":"2872","DOI":"10.1021\/ct100326h","article-title":"Computational Thermochemistry:\nScale Factor Databases and Scale Factors for Vibrational Frequencies\nObtained from Electronic Model Chemistries","volume":"6","author":"Alecu","year":"2010","journal-title":"J. Chem. Theory. Comput."},{"issue":"3","key":"2026101310072513000_cit29","doi-asserted-by":"publisher","first-page":"597","DOI":"10.1021\/ct900566x","article-title":"Efficient Diffuse\nBasis Sets for Density Functional\nTheory","volume":"6","author":"Papajak","year":"2010","journal-title":"J. Chem. Theory Comput."},{"issue":"27","key":"2026101310072513000_cit30","doi-asserted-by":"publisher","first-page":"3172","DOI":"10.1039\/B517914A","article-title":"Advances in Methods and Algorithms in a Modern Quantum\nChemistry Program Package.Advances in methods and algorithms in a\nmodern quantum chemistry program package","volume":"8","author":"Shao","year":"2006","journal-title":"Phys.\nChem. Chem. Phys."},{"issue":"10","key":"2026101310072513000_cit31","doi-asserted-by":"publisher","first-page":"5273","DOI":"10.1021\/acs.jctc.8b00529","article-title":"Tinker\n8: Software Tools for Molecular Design","volume":"14","author":"Rackers","year":"2018","journal-title":"J. Chem.\nTheory. Comput."},{"issue":"2","key":"2026101310072513000_cit32","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1021\/ci400640v","article-title":"Quantum Mechanical\/Molecular\nMechanical Study of Catalytic Mechanism\nand Role of Key Residues in Methylation Reactions Catalyzed by Dimethylxanthine\nMethyltransferase in Caffeine Biosynthesis","volume":"54","author":"Yue","year":"2014","journal-title":"J. Chem. Inf. Model."},{"key":"2026101310072513000_cit33","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1016\/j.jare.2022.03.014","article-title":"Identification of oxidosqualene cyclases associated with saponin\nbiosynthesis from Astragalus membranaceus reveals a conserved motif\nimportant for catalytic function","volume":"43","author":"Chen","year":"2023","journal-title":"J. Adv. Res."},{"key":"2026101310072513000_cit34","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/j.sbi.2016.05.019","article-title":"Squalene-hopene\ncyclases\u2500evolution, dynamics and catalytic scope","volume":"41","author":"Syre\u0301n","year":"2016","journal-title":"Curr. Opin. Struct. Biol."},{"key":"2026101310072513000_cit35","doi-asserted-by":"publisher","DOI":"10.1016\/j.plipres.2024.101287","article-title":"Plant terpenoid biosynthetic\nnetwork and its multiple layers of regulation","volume":"95","author":"Bergman","year":"2024","journal-title":"Prog. Lipid Res."},{"issue":"1","key":"2026101310072513000_cit36","doi-asserted-by":"publisher","first-page":"195","DOI":"10.1007\/s00253-024-13030-8","article-title":"Engineering\namino acid residues of pentacyclic triterpene synthases for improving\nthe activity","volume":"108","author":"Guo","year":"2024","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"2026101310072513000_cit37","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2025.141340","article-title":"The key structure and\nself-stabilization mechanism of water-soluble interfacial squalene-hopene\ncyclase","volume":"305","author":"Chen","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"issue":"10","key":"2026101310072513000_cit38","doi-asserted-by":"publisher","first-page":"2658","DOI":"10.1002\/anie.201207842","article-title":"The Importance\nof Additive and Non-Additive Mutational Effects in Protein Engineering","volume":"52","author":"Reetz","year":"2013","journal-title":"Angew. Chem., Int. Ed."},{"issue":"20","key":"2026101310072513000_cit39","doi-asserted-by":"publisher","first-page":"2864","DOI":"10.1021\/acs.biochem.6b00343","article-title":"Probing\nthe Role of Active Site Water\nin the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene\nSynthase","volume":"55","author":"Chen","year":"2016","journal-title":"Biochemistry"}],"container-title":["Journal of Chemical Theory\nand Computation"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jctc.6c01306","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jctcce\/article-pdf\/22\/19\/10465\/68563465\/acs.jctc.6c01306.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jctcce\/article-pdf\/22\/19\/10465\/68563465\/acs.jctc.6c01306.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T19:07:11Z","timestamp":1791918431000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jctcce\/article\/22\/19\/10465\/5433250\/Modeling-the-Impact-of-Active-Site-Mutations-on"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,19]]},"references-count":39,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,9,19]]},"published-print":{"date-parts":[[2026,10,13]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jctc.6c01306","relation":{},"ISSN":["1549-9618","1549-9626"],"issn-type":[{"value":"1549-9618","type":"print"},{"value":"1549-9626","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,19]]}}}