2026-10-14T07:48:08Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33942560 2026-09-19T14:10:48Z category_1 category_4 category_13 category_14 category_272 category_873 category_734 category_931 category_61 category_135 portal_63 item_type_3 month_year_09_2026

Modeling the Impact of Active Site Mutations on the Quenching Fate and Cyclization Depth of a Rare Oxidosqualene Cyclase Chenxu Liu (6032342) Shun Liang (42139) Ying Zheng (170527) Feng Wang (44414) Jiaqi Jiao (23832252) Bing Li (43671) JinCai Liang (25076117) Zhongqiu Liu (50019) Lixin Duan (593581) Ruibo Wu (1374138) Fan Zhang (46132) Biophysics Biochemistry Genetics Molecular Biology Environmental Sciences not elsewhere classified Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Information Systems not elsewhere classified Developmental Biology Computational Biology robust theoretical basis panax ginseng </ integrating multiscale qm induces catalytic promiscuity hydroxylated product profile homologous dammarenediol synthase general base governing form dammarenediol ii exhibiting absolute specificity driven enzyme reshaping diverse polycyclic triterpenoids convert linear 2 comprehensive mechanistic framework artemisia argyi </ relaxing steric constraints driven functional reprogramming yield dammarenediol ii active site mutations ultimate quenching mechanism steric cavity functional loci quenching network quenching fate yield tricyclic triterpene synthases transfer network tetracyclic products terminal deprotonation synthetic biology study provides starkly contrasts simulations revealed reaction cascade rare osc pure deprotonation produce dammara precise control pgdds ), pentacyclic lupeol mm simulations investigate aaosc hydroxylation pathway fundamental challenge favors hydroxylation dramatically alter directed mutagenesis cyclization depth chemical trajectory 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 <i>Artemisia argyi</i> exhibiting absolute specificity for pure deprotonation to produce dammara-20,24-dien-3β-ol. This starkly contrasts with the homologous dammarenediol synthase from <i>Panax ginseng</i> (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. 2026-09-19T00:00:00Z Dataset Dataset 10.1021/acs.jctc.6c01306.s001 https://figshare.com/articles/dataset/Modeling_the_Impact_of_Active_Site_Mutations_on_the_Quenching_Fate_and_Cyclization_Depth_of_a_Rare_Oxidosqualene_Cyclase/33942560 CC BY-NC 4.0