Tunnel Engineering for Modulating The Substrate Preference in Decarbonylase P450BsβHI

Abstract

An active site normally locates inside of enzymes, substrates should go through the tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450_BsβHI (Q85H/ V170I) derived from hydroxylase P450_Bsβ from Bacillus subtilis was chosen as study model, which is reported as a potential decarbonylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarbonylase activity of P450_BsβHI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.