Macromolecular Structural Biology Lab, Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, Telangana 502285, India.
National Institute of Pharmaceutical Education and Research (NIPER), NH-44, Balanagar, Hyderabad 500037, India.
ACS Chem Biol. 2022 Feb 18;17(2):266-280. doi: 10.1021/acschembio.1c00500. Epub 2022 Jan 18.
Enzyme-based synthetic chemistry provides a green way to synthesize industrially important chemical scaffolds and provides incomparable substrate specificity and unmatched stereo-, regio-, and chemoselective product formation. However, using biocatalysts at an industrial scale has its challenges, like their narrow substrate scope, limited stability in large-scale one-pot reactions, and low expression levels. These limitations can be overcome by engineering and fine-tuning these biocatalysts using advanced protein engineering methods. A detailed understanding of the enzyme structure and catalytic mechanism and its structure-function relationship, cooperativity in binding of substrates, and dynamics of substrate-enzyme-cofactor complexes is essential for rational enzyme engineering for a specific purpose. This Review covers all these aspects along with an in-depth categorization of various industrially and pharmaceutically crucial bisubstrate enzymes based on their reaction mechanisms and their active site and substrate/cofactor-binding site structures. As the bisubstrate enzymes constitute around 60% of the known industrially important enzymes, studying their mechanism of actions and structure-activity relationship gives significant insight into deciding the targets for protein engineering for developing industrial biocatalysts. Thus, this Review is focused on providing a comprehensive knowledge of the bisubstrate enzymes' structure, their mechanisms, and protein engineering approaches to develop them into industrial biocatalysts.
基于酶的合成化学为合成工业上重要的化学支架提供了一种绿色的方法，并且提供了无与伦比的底物特异性和无与伦比的立体、区域和化学选择性产物形成。然而，在工业规模上使用生物催化剂存在一些挑战，例如它们的底物范围狭窄、在大规模一锅反应中的稳定性有限以及表达水平低。这些限制可以通过使用先进的蛋白质工程方法对这些生物催化剂进行工程设计和微调来克服。详细了解酶的结构和催化机制及其结构-功能关系、底物结合的协同性以及底物-酶-辅因子复合物的动力学对于针对特定目的进行合理的酶工程至关重要。本综述涵盖了所有这些方面，并根据其反应机制以及其活性位点和底物/辅因子结合位点结构对各种工业和制药关键的双底物酶进行了深入分类。由于双底物酶约占已知工业上重要酶的 60%，因此研究它们的作用机制和结构-活性关系可以深入了解决定蛋白质工程目标以开发工业生物催化剂的情况。因此，本综述重点介绍了双底物酶的结构、它们的机制以及蛋白质工程方法的综合知识，以将它们开发为工业生物催化剂。
