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首页> 外文期刊>ACS catalysis >Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome
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Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome

机译：实验和仿真揭示了功能塑性区域的突变如何引导植物单萜合酶产品结果

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Monoterpenes (C10 isoprenoids) are a structurally diverse group of natural compounds that are attractive to industry as flavors and fragrances. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyze high-energy cyclization reactions involving unstable carbocation intermediates. Efforts toward producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclization type. We used available mutagenesis data to show that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Consensus sequences for three such plasticity regions were introduced in different mTC/S with increasingly complex cyclization cascades, including the model enzyme limonene synthase (LimS). In all three mTC/Ss studied, mutations in the first two regions mostly give rise to products that result from premature quenching of the linalyl or a-terpinyl cations, suggesting that both plasticity regions are involved in the formation and stabilization of cations early in the reaction cascade. A LimS variant with mutations in the second region (S454G, C4S7V, M4581), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclization is not due to compression of the C2-C7 distance in the a-terpinyl cation but is the result of an increased distance between C8 of the alpha-terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the effect of mutations can only be obtained using integrated experimental and computational approaches and will aid the design of altered mTC/S activities toward clean monoterpenoid products.
机译：单波通（C10异戊二烯）是一种结构各种各样的天然化合物，对工业具有味道和香料具有吸引力。单波通由单个线性基材，由一种称为单萜烯酶/合成酶（MTC / SS）的酶的酶产生，该酶催化涉及不稳定的碳结构中间体的高能量环化反应。通过生物分析或代谢工程生产单口萜烯的努力通常导致由于MTC / SS的高分支反应机制的性质而导致多种产品的形成。通过缺乏有源部位序列和环化类型之间的相关性阻碍了MTC / SS的合理工程。我们使用可用的诱变数据来表明涉及产品结果的氨基酸在MTC / S家族内聚集和空间上保守。具有三种这种可塑性区域的共有序列在不同的MTC / s中引入了越来越复杂的环化级联，包括模型酶柠檬烯合酶（LIMS）。在研究的所有三种MTC / SS中，前两个地区的突变主要导致Linalyl或萜烯阳离子过早猝灭导致的产品，这表明两种塑性区域都涉及阳离子早期的形成和稳定性反应级联。具有第二区域突变的LIMS变体（S454G，C4S7V，M4581），主要生产的双环产品主要。 QM / mm MD模拟显示第二环化不是由于α-萜烯阳离子中的C2-C7距离的压缩，而是α-萜烯基阳离子和两个推定碱（W324）之间的C8之间的距离增加的结果（W324， H579）位于活性位点的另一边，通过去质子化预防早期终止。这些见解只能使用综合的实验和计算方法获得突变的效果，并将有助于设计改变的MTC / S活性对清洁的单萜类化产品。

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