In recent years, the engineering of flexible loops to
improve enzyme
properties has gained attention in biocatalysis. Herein, we report
a loop engineering strategy to improve the stability of the substrate
access tunnels, which reveals the molecular mechanism between loops
and tunnels. Based on the dynamic tunnel analysis of CYP116B3, five
positions (A86, T91, M108, A109, T111) in loops B-B′ and B′-C
potentially affecting tunnel frequent occurrence were selected and
subjected to simultaneous saturation mutagenesis. The best variant
8G8 (A86T/T91L/M108N/A109M/T111A) for the dealkylation of 7-ethoxycoumarin
and the hydroxylation of naphthalene was identified with considerably
increased activity (134-fold and 9-fold) through screening. Molecular
dynamics simulations showed that the reduced flexibility of loops
B-B′ and B′-C was responsible for increasing the stability
of the studied tunnel. The redesign of loops B-B′ and B′-C
surrounding the tunnel entrance provides loop engineering with a powerful
and likely general method to kick on/off the substrate/product transportation.
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, P450BsβHI (Q85H/V170I) derived from hydroxylase P450Bsβ 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 P450BsβHI. 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.
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