Site-Saturated Mutagenesis of Histidine 234 of Saccharomyces cerevisiae Oxidosqualene-Lanosterol Cyclase Demonstrates Dual Functions in Cyclization and Rearrangement Reactions
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- 作者：Tung-Kung Wu ; Yuan-Ting Liu ; Cheng-Hsiang Chang ; Mei-Ting Yu ; and Hisng-Ju Wang
- 刊名：Journal of the American Chemical Society
- 出版年：2006
- 出版时间：May 17, 2006
- 年：2006
- 卷：128
- 期：19
- 页码：6414 - 6419
- 全文大小：147K
- 年卷期：v.128,no.19(May 17, 2006)
- ISSN：1520-5126
文摘
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 activityand to determine its effect on the oxidosqualene cyclization/rearrangement reaction. Two novel intermediates,(13
H)-isomalabarica-14(26),17E,21-trien-3
-ol and protosta-20,24-dien-3-ol, isolated from ERG7H234Xmutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikovcation 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 lanosterolformation to either protosta-12,24-dien-3-ol or parkeol production. Finally, the repeated observation ofmultiple abortive and/or alternative cyclization/arrangement products from various ERG7H234X mutantsdemonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affectboth the stability of the cation-
interaction and generate product diversity.
H)-isomalabarica-14(26),17E,21-trien-3-ol and protosta-20,24-dien-3-ol, isolated from ERG7H234Xmutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikovcation 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 lanosterolformation to either protosta-12,24-dien-3-ol or parkeol production. Finally, the repeated observation ofmultiple abortive and/or alternative cyclization/arrangement products from various ERG7H234X mutantsdemonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affectboth the stability of the cation- interaction and generate product diversity.