title: Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions.
abstract: 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, (13alphaH)-isomalabarica-14(26),17E,21-trien-3beta-ol and protosta-20,24-dien-3beta-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-3beta-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.
authors: Department of Biological Science and Technology, National Chiao Tung University, 300, Hsin-Chu, Taiwan (Republic of China). tkwmll@mail.nctu.edu.tw
authors: Tung-Kung
authors: TK
authors: Wu
authors: Yuan-Ting
authors: YT
authors: Liu
authors: Cheng-Hsiang
authors: CH
authors: Chang
authors: Mei-Ting
authors: MT
authors: Yu
authors: Hisng-Ju
authors: HJ
authors: Wang