Tunnel engineering for modulating the substrate preference in cytochrome P450BsβHI

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(Bs beta)HI (Q85H/V170I) derived from hydroxylase P450(Bs beta) 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(Bs beta)HI. The finally obtained Bs beta HI-F79A variant had a 15.2-fold improved conversion for palmitic acid; Bs beta HI-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.

Automated summary

The study demonstrates how tunnel engineering can modulate substrate preference and improve decarboxylation activity of enzymes, resulting in significantly increased conversion rates for specific fatty acids.