Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel
Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel
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DOI：
10.1128/aem.01817-16
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发表时间：
2016-09
影响因子：
4.4
通讯作者：
Xiaodan Yan;Jianjun Wang;Yu Sun;Junge Zhu;Sheng Wu
中科院分区：
文献类型：
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作者：
Xiaodan Yan;Jianjun Wang;Yu Sun;Junge Zhu;Sheng Wu
摘要混杂酶通常被认为是进化出具有更特异甚至新的催化活性的后代酶的起点，是产生新的生物学功能的分子基础。Mhg是一种典型的α/β折叠水解酶，以前报道同时具有γ-内酰胺酶和过水解酶活性。然而，尽管具有高度的结构相似性，并共享一个相同的催化三联体与广泛研究的酯酶从荧光假单胞菌，这种酶没有显示出任何酯酶活性。分子对接和序列分析表明，结合口袋的进入可能在阻断入口通道中起作用，阻止酯类化合物进入口袋。通过仅用一个或两个氨基酸取代工程化入口通道，我们成功地获得了Mhg的五种酯酶变体。这些变体表现出非常广泛的底物接受性，不仅水解经典的对硝基苯酚酯，而且水解广泛用作药物中间体的各种类型的手性酯。Mhg入口通道的233位点通过调节通道的大小和形状在调节三种催化活性中起着关键作用，该位点的不同氨基酸取代促进不同的活性。值得注意的是，具有L233 G突变的变体是一种非常特异的酯酶，没有任何γ-内酰胺酶和过水解酶活性。考虑到氨基酸的保守性和差异性，该位点可能是未来蛋白质工程的关键目标。此外，我们证明了工程入口隧道是一种有效的策略，以调节酶的催化能力。混杂酶可以作为新催化活性进化的起点，从而为新生物功能的产生提供分子基础。在这项研究中，我们确定了一个关键的氨基酸残基（Leu 233）在一个混杂的酶，Mhg的底物隧道的入口。我们发现，用较小的氨基酸如Gly、Ala、Ser或Pro取代该残基赋予该酶新的酯酶活性。该位点上的不同氨基酸可以促进不同的催化活性。这些发现在α/β折叠水解酶亚组（包括Mhg）中具有普遍意义。此外，我们证明了工程入口隧道是一种有效的策略，以发展新的酶催化能力。我们的研究对酶催化混杂性的调控和蛋白质工程方法的发展具有重要意义。
ABSTRACT Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical α/β fold hydrolase, was previously reported to have both γ-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any γ-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities. IMPORTANCE Promiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of α/β fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies.