Liu Ke, Jiang Ling, Ma Shuang, Song Zhongdi, Wang Lun, Zhang Qunfeng, Xu Renhao, Yang Lirong, Wu Jianping, Yu Haoran
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, Zhejiang, China.
ZJU-Hangzhou Global Scientific and Technological Innovation Centre, Hangzhou, 311200, Zhejiang, China.
Bioresour Bioprocess. 2023 Dec 11;10(1):92. doi: 10.1186/s40643-023-00712-w.
Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.
氨酰 - tRNA合成酶（aaRS）是遗传密码扩展（GCE）的核心组件，GCE是一种强大的技术，能够将非天然氨基酸（ncAA）掺入蛋白质中。具有多特异性的aaRS可用于在不进化新的正交aaRS/tRNA对的情况下将额外的ncAA掺入蛋白质中，因此为探究酶机制或扩展蛋白质功能提供了有用的工具。来自马氏甲烷八叠球菌（MmPylRS）的吡咯赖氨酸 - tRNA合成酶变体（N346A/C348A）表现出广泛的底物范围，可接受40多种苯丙氨酸衍生物。然而，对于大多数底物，掺入效率较低。在此，构建了一种MbPylRS（N311A/C313A）变体，其显示出比同源的MmPylRS（N346A/C348A）更高的ncAA掺入效率。接下来，通过先前鉴定的单个变体的贪婪组合对MbPylRS（N311A/C313A）的N端进行工程改造，产生了一种IPE（N311A/C313A/V31I/T56P/A100E）变体，其对各种ncAA的活性显著提高。然后测试了IPE对43种新型ncAA的活性，其中16种被鉴定为该变体可接受。因此，该变体总共可将近60种ncAA掺入蛋白质中。利用该变体，然后将8种不同的ncAA掺入一种镧系元素依赖性醇脱氢酶PedH中。苯乳酸的掺入使PedH对甲醇的催化效率提高了1.8倍，表明在酶工程中修饰蛋白质主链的作用。O - 叔丁基 - L - 酪氨酸的掺入通过影响底物与蛋白质之间的相互作用改变了PedH的对映选择性。酶学表征和分子动力学模拟揭示了ncAA影响PedH催化的机制。本研究提供了一种具有高活性和底物混杂性的PylRS变体，这增加了GCE在酶机制阐释和工程中的实用性。
