Increasing complexity of a diterpene synthase reaction with a single residue switch
Increasing complexity of a diterpene synthase reaction with a single residue switch
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DOI：
10.1021/ja710524w
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发表时间：
2008-04-23
影响因子：
15
通讯作者：
Peters, Reuben J.
中科院分区：
文献类型：
--
作者：
Morrone, Dana;Xu, Meimei;Peters, Reuben J.
萜烯脱氢酶通常催化复杂的反应，涉及一系列复杂的碳阳离子中间体。所产生的通常是环状的结构提供了最初的烃框架，这些框架构成了大量萜类天然产物（已知> 50，000种）惊人的结构多样性的基础，并且这些酶通常介导其特定生物合成途径中的关键步骤。因此，萜烯糖苷酶如何指定产物产物已经引起了大量的关注。在以前的工作中，我们已经表明，在二萜合酶活性位点内的特定位置处突变引入羟基可以“短路”复杂的环化和/或重排反应，导致产生“更简单”的二萜。在这里，我们表明，匡威的变化，取代的一个谎言的Thr在相关的位置在一个天然的海松二烯合酶，导致反应的复杂性急剧增加。产物结果从三环海松二烯转变为重排的四环，aphidicol-15-烯。因此，在该位置处的残基的性质充当产物结果的真正开关。此外，脂肪族残基取代的能力，使一个更复杂的反应强调了底物构象的重要性，由一个很大程度上惰性的活性位点。此外，这种单一的残基开关的产品结果所例示的二萜类脱氢酶的深刻的可塑性是一致的筛选/多样性为导向的天然产物代谢的假设。
Terpene synthases often catalyze complex reactions involving intricate series of carbocation intermediates The resulting, generally cyclical, structures provide initial hydrocarbon frameworks that underlie the astonishing structural diversity of the enormous class of terpenoid natural products (>50,000 known), and these enzymes often mediate the committed step in their particular biosynthetic pathway. Accordingly, how terpene synthases specify product outcome has drawn a great deal of attention. In previous work, we have shown that mutational introduction of a hydroxyl group at specific positions within diterpene synthase active sites can "short circuit" complex cyclization and/or rearrangement reactions, resulting in the production of "simpler" diterpenes. Here we demonstrate that the converse change, substitution of an lie for Thr at the relevant position in a native pimaradiene synthase, leads to a dramatic increase in reaction complexity. Product outcome is shifted from the tricyclic pimaradiene to a rearranged tetracycle, aphidicol-15-ene. Thus, the nature of the residue at this position acts as a true switch for product outcome. In addition, the ability of aliphatic residue substitution to enable a more complex reaction emphasizes the importance of substrate conformation imposed by a largely inert active site. Furthermore, the profound plasticity of diterpene synthases exemplified by this single residue switch for product outcome is consistent with the screening/diversity-oriented hypothesis of natural products metabolism.