Xiang Chao, Ce Yu-Ke, Xue Ya-Ping, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China.
Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China.
Biotechnol Lett. 2025 Mar 18;47(2):35. doi: 10.1007/s10529-025-03577-5.
In enzyme engineering, a lot of studies have focused on engineering the active site to broaden substrate specificity or enhance transaminase activity; however, relatively little is known about the mechanisms by which substrates are recognized and enter the binding pocket. Transaminases play a crucial role in the synthesis of chiral amines due to their exceptional stereoselectivity and catalytic efficiency. In this study, we explored how the pedal-like loop at the active site influences (R)-transaminase (ATA) activity and substrate recognition by modulating the substrate channel. The pedal-like loop at the active site was swapped with loops from other well-characterized transaminases, and the best-performing variant exhibited a 5.2-fold increase in activity toward (R)-phenylethylamine ((R)-PEA) and an 11.8-fold increase in activity toward isopropylamine (IPA). Additionally, some variants showed significant changes in substrate preference. Homology modeling and molecular docking analysis provided compelling evidence that the pedal-like loop is a critical determinant of both substrate recognition and catalytic activity in (R)-ATA.
在酶工程中，许多研究都集中在对活性位点进行工程改造，以拓宽底物特异性或提高转氨酶活性；然而，对于底物被识别并进入结合口袋的机制，人们了解得相对较少。转氨酶因其卓越的立体选择性和催化效率，在手性胺的合成中起着至关重要的作用。在本研究中，我们探究了活性位点处的踏板状环如何通过调节底物通道来影响（R）-转氨酶（ATA）活性和底物识别。将活性位点处的踏板状环与其他特征明确的转氨酶的环进行交换，表现最佳的变体对（R）-苯乙胺（（R）-PEA）的活性提高了5.2倍，对异丙胺（IPA）的活性提高了11.8倍。此外，一些变体在底物偏好上表现出显著变化。同源建模和分子对接分析提供了有力证据，表明踏板状环是（R）-ATA中底物识别和催化活性的关键决定因素。
