In Silico Based Engineering Approach to Improve Transaminases for the Conversion of Bulky Substrates
In Silico Based Engineering Approach to Improve Transaminases for the Conversion of Bulky Substrates
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DOI：
10.1021/acscatal.8b03900
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发表时间：
2018-10
期刊：
影响因子：
12.9
通讯作者：
M. Voss;D. Das;M. Genz;Anurag Kumar;N. Kulkarni;J. Kustosz;Pravin Kumar;U. Bornscheuer;M. Höhne
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文献类型：
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作者：
M. Voss;D. Das;M. Genz;Anurag Kumar;N. Kulkarni;J. Kustosz;Pravin Kumar;U. Bornscheuer;M. Höhne
蛋白质工程通常用于根据过程的需要定制底物特异性，对映体选择性或酶的稳定性。在合理的工程方法中，分子对接和分子动力学模拟常用于比较野生型和酶变体的过渡状态。除了通过突变影响过渡态能量外，底物的进入及其在活性位点（Michaelis复合体）的定位也经常被研究，形成底物进入通道的残基的突变对活性和选择性有深远的影响。在本研究中，我们将这种隧道方法的优势与MD结合起来，然后进行半经验QM计算，从而确定有益的位置并对可能的变体进行计算机筛选。我们在紫色色杆菌胺转氨酶的底物范围向立体要求底物的扩展中举例说明了这一策略。两个双突变体（F88L/C418(G/L)）…
Protein engineering is often applied to tailor substrate specificity, enantioselectivity, or stability of enzymes according to the needs of a process. In rational engineering approaches, molecular docking and molecular dynamics simulations are often used to compare transition states of wild-type and enzyme variants. Besides affecting the transition state energies by mutations, the entry of the substrate and its positioning in the active site (Michaelis complex) is also often studied, and mutagenesis of residues forming the substrate entry tunnel can have a profound impact on activity and selectivity. In this study, we combine the strengths of such a tunnel approach with MD followed by semiempirical QM calculations that allow the identification of beneficial positions and an in silico screening of possible variants. We exemplify this strategy in the expansion of the substrate scope of Chromobacterium violaceum amine transaminase toward sterically demanding substrates. Two double mutants (F88L/C418(G/L)) pr...