Browsing by Author "Yu, Mei-Ting"

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Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions
(2006-05-17)
Wu, Tung-Kung
;
Liu, Yuan-Ting
;
Chang, Cheng-Hsiang
;
Yu, Mei-Ting
;
Wang, Hisng-Ju
Site-saturated mutagenesis experiments were carried out on the His234 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) to characterize its functional role in ERG7 activity and to determine its effect on the oxidosqualene cyclization/ rearrangement reaction. Two novel intermediates, (13 alpha H)-isomalabarica-14(26), 17E,21-trien-3 beta-ol and protosta-20,24-dien-3 beta-ol, isolated from ERG7(H234X) mutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikov cation and protosteryl cation that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. In addition, we obtained mutants that showed a complete change in product specificity from lanosterol formation to either protosta-12,24-dien-3 beta-ol or parkeol production. Finally, the repeated observation of multiple abortive and/or alternative cyclization/ arrangement products from various ERG7(H234X) mutants demonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affect both the stability of the cation-pi interaction and generate product diversity.