Energy and conformation determine the enantioselectivity of enzyme

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Enantioselectivity of biocatalysts is a crucial property utilized for the synthesis of stereo-specific products. Owing to its complex mechanism, the development of precise and simple way to understand and alter enantioselectivity has been a significant pursuit and challenge for decades. In this study, an accurate calculation by MM/PBSA method was applied to investigate the binding free energy of R- and S-type substrates with enzyme. Meanwhile, a simple distance standard was developed to evaluates the binding conformation. Furthermore, we proposed that the binding free energy and binding conformation had significant effect on enzyme enantioselectivity simultaneously, and played pivotal role in Km and kcat, respectively. A well-studied, important lipase LipK107, and both positive and negative mutants were explored to validate our finding. Compared with WT, the mutations indeed resulted in the alteration of both binding energy and conformation, which collectively led to the changes of enzyme enantioselectivity. Our work indicated that rational enzyme engineer should attach great importance on both the binding free energy and the binding conformation of substrate and enzyme. Besides, since the binding energy and conformation could be calculated precisely, a promising large-scale in silico design strategy could be applied to gain various enzymes with outstanding characteristics.