Tunnel Engineering for Modulating the Substrate Preference in Cytochrome P450BsβHI


Congratulations Shuaiqi Meng on his recent publication!

Enzymatic oxidative decarboxylation by cytochrome P450s represents one of the simplest strategies for α-alkenes synthesis. In this study, we reshaped the substrate access tunnels of cytochrome P450BsβHI for improving the decarboxylation activity towards long chain fatty acids. Compared with the natural substrate lauric acid, long-chain fatty acids require wider substrate access tunnels to reach the P450BsβHI active site from the solvent environment. Hence, a dynamic tunnel analysis on P450BsβHI was conducted and two phenylalanine residues (F79 and F173) were identified as limitation of the tunnel radius. After replacement of the phenylalanine residues by smaller amino acids, variants showed increased decarboxylation activity towards long chain fatty acids. Finally, the obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; the BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. This study shows that identifying and engineering key residues lining the access tunnels may be a valuable and efficient strategy for improving the performance of enzymes with buried active sites.

This study was funded by the National Natural Science Foundation of China (grant number 31961133017, 21978017, 21978020, 21861132017). These grants are part of MIX-UP, a joint NSFC and EU H2020 collaboration (No 870294). The Fundamental Research Funds for the Central Universities XK1802-8. Shuaiqi Meng was supported by a Ph.D. scholarship from the China Scholarship Council (CSC No. 201906880011).

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Meng S, An R, Li Z, Schwaneberg U, Ji Y, Davari MD, Wang F, Wang M, Qin M, Nie K, Liu L. Tunnel engineering for modulating the substrate preference in cytochrome P450BsβHI. BIOB. 2021 Bioresour. Bioprocess. 8, 26 (2021). https://doi.org/10.1186/s40643-021-00379-1.

  Enzyme and substrate flow Copyright: © BioMed Central Ltd Engineering of substrate access tunnels is an effective strategy for rational modulation of substrate preference in cytochrome P450s.