Conformational Change and Ligand Binding in the Aristolochene Synthase Catalytic Cycle
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- 作者：Marc W. van der Kamp ; Jitnapa Sirirak ; Jolanta 呕urek ; Rudolf K. Allemann ; Adrian J. Mulholland
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：November 12, 2013
- 年：2013
- 卷：52
- 期：45
- 页码：8094-8105
- 全文大小：720K
- 年卷期：v.52,no.45(November 12, 2013)
- ISSN：1520-4995
文摘
Terpene synthases are potentially useful biocatalysts for the synthesis of valuable compounds, such as anticancer drugs and antibiotics. The design of altered activities requires better knowledge of their mechanisms, for example, an understanding of the complex conformational changes that are part of their catalytic cycle, how they are coordinated, and what drives them. Crystallographic studies of the sesquiterpene synthase artistolochene synthase have led to a proposed sequence of ligand binding and conformational change but have provided only indirect insight. Here, we have performed extensive molecular dynamics simulations of multiple enzyme鈥搇igand complexes (over 2 渭s in total). The simulations provide clear evidence of what drives the conformational changes required for reaction. They support a picture in which the substrate farnesyl diphosphate binds first, followed by three magnesium ions in sequence, and, after reaction, the release of aristolochene and two magnesium ions followed by the final magnesium ion and diphosphate. Binding of farnesyl diphosphate leads to an increased level of sampling of open conformations, allowing the first two magnesium ions to bind. The closed enzyme conformation is maintained with a diphosphate moiety and two magnesium ions bound. The open-to-closed transition reduces flexibility around the active site entrance, partly through a lid closing over it. The simulations with all three magnesium ions and farnesyl diphosphate bound provide, for the first time, a realistic model of the Michaelis complex involved in reaction, which is inaccessible to experimental structural studies. These insights could help with the design of altered activities in a range of terpene synthases.