title: Protein engineering of oxidosqualene-lanosterol cyclase into triterpene monocyclase.
abstract: A computational modeling/protein engineering approach was applied to probe H234, C457, T509, Y510, and W587 within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7), which spatially affects the C-10 cation of lanosterol formation. Substitution of Trp587 to aromatic residues supported the "aromatic hypothesis" that the π-electron-rich pocket is important for the stabilization of electron-deficient cationic intermediates. The Cys457 to Gly and Thr509 to Gly mutations disrupted the pre-existing H-bond to the protonating Asp456 and the intrinsic His234 : Tyr510 H-bond network, respectively, and generated achilleol A as the major product. An H234W/Y510W double mutation altered the ERG7 function to achilleol A synthase activity and generated achilleol A as the sole product. These results support the concept that a few-ring triterpene synthase can be derived from polycyclic cyclases by reverse evolution, and exemplify the power of computational modeling coupled with protein engineering both to study the enzyme's structure-function-mechanism relationships and to evolve new enzymatic activity.
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, 300, Hsin-Chu, Taiwan, Republic of China.
authors.forename: Cheng-Hsiang
authors.initials: CH
authors.lastname: Chang
authors.forename: Hao-Yu
authors.initials: HY
authors.lastname: Wen
authors.forename: Wen-Shiang
authors.initials: WS
authors.lastname: Shie
authors.forename: Ching-Ting
authors.initials: CT
authors.lastname: Lu
authors.forename: Meng-Erh
authors.initials: ME
authors.lastname: Li
authors.forename: Yuan-Ting
authors.initials: YT
authors.lastname: Liu
authors.forename: Wen-Hsuan
authors.initials: WH
authors.lastname: Li
authors.forename: Tung-Kung
authors.initials: TK
authors.lastname: Wu
mesh_terms: D019943:Amino Acid Substitution; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014315:Triterpenes / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014315:Triterpenes; D019751:Intramolecular Transferases; C021924:lanosterol synthase