標題: Protein engineering of oxidosqualene-lanosterol cyclase into triterpene monocyclase
作者: Chang, Cheng-Hsiang
Wen, Hao-Yu
Shie, Wen-Shiang
Lu, Ching-Ting
Li, Meng-Erh
Liu, Yuan-Ting
Li, Wen-Hsuan
Wu, Tung-Kung
生物科技學系
Department of Biological Science and Technology
公開日期: 2013
摘要: 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 pi-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.
URI: http://hdl.handle.net/11536/22457
http://dx.doi.org/10.1039/c3ob40493e
ISSN: 1477-0520
DOI: 10.1039/c3ob40493e
期刊: ORGANIC & BIOMOLECULAR CHEMISTRY
Volume: 11
Issue: 25
起始頁: 4214
結束頁: 4219
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