Tunnel engineering for modulating the substrate preference in cytochrome P450_BsβHI

Abstract

An active site is normally located inside enzymes, hence substrates should go through a 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 the study model, which is reported as a potential decarboxylase. 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 decarboxylation 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.

Keywords

Tunnel engineering / Substrate preference / Cytochrome P450_BsβHI / α-Alkene biosynthesis / Rational design

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Funding

National Natural Science Foundation of China(31961133017)

European Union’s Horizon 2020(870294)