Sparse Epistatic Patterns in the Evolution of Terpene Synthases
Sparse Epistatic Patterns in the Evolution of Terpene Synthases
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DOI：
10.1093/molbev/msaa052
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发表时间：
2020-07-01
影响因子：
10.7
通讯作者：
Morozov, Alexandre, V
中科院分区：
文献类型：
--
作者：
Ballal, Aditya;Laurendon, Caroline;Morozov, Alexandre, V
我们探讨了一个主要的萜烯脱氢酶家族的酶活性和特异性的序列决定因素。这个家族中的大多数酶催化产生环萜类化合物的反应，环萜类化合物广泛用于植物和昆虫的各种生物过程，如防御，通讯和共生。为了分析萜烯环化出现的分子机制，我们对（E）-β-法呢烯合酶（一种催化线性烃链产生的青蒿酶）周围的突变空间进行了深入研究。在我们的合成库中的每个突变酶的特征在于生物化学，和所得的反应速率数据被用作输入的Michaelis-Menten模型的酶动力学，其中自由能表示为一个氨基酸的贡献和两个氨基酸的耦合的总和。我们的模型预测测得的反应速率具有高精度，并产生自由能景观，其特征在于相对较少的耦合项。因此，Michaelis-Menten自由能景观具有简单，可解释的结构，并表现出很少的上位性。我们还开发了生物物理适应性模型，该模型基于以下假设：高度适应的酶已经进化为最大化正确产物的输出，例如环状产物或感兴趣的特定产物，同时最小化副产物的输出。这种方法导致非线性适应度景观，这是相当多的上位性。总的来说，我们的实验和计算框架提供了集中的表征新的酶功能的进化出现的背景下，自然发生的酶周围的序列空间的微进化探索。
We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes-complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-beta-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon chain. Each mutant enzyme in our synthetic libraries was characterized biochemically, and the resulting reaction rate data were used as input to the Michaelis-Menten model of enzyme kinetics, in which free energies were represented as sums of one-amino-acid contributions and two-amino-acid couplings. Our model predicts measured reaction rates with high accuracy and yields free energy landscapes characterized by relatively few coupling terms. As a result, the Michaelis-Menten free energy landscapes have simple, interpretable structure and exhibit little epistasis. We have also developed biophysical fitness models based on the assumption that highly fit enzymes have evolved to maximize the output of correct products, such as cyclic products or a specific product of interest, while minimizing the output of byproducts. This approach results in nonlinear fitness landscapes that are considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of evolutionary emergence of novel enzymatic functions in the context of microevolutionary exploration of sequence space around naturally occurring enzymes.