Modulating the coupling efficiency of P450 BM3 by controlling water diffusion through access tunnel engineering

  Shuaiqi Meng and Ju Yi Copyright: © BIO VI

Meng, S., Ji, Y., Liu, L., Davari, M. D., Schwaneberg, U., ChemSusChem,

Introduction of hydrophobic residues to substrate access tunnels increased the catalytic efficiency and coupling efficiency of P450 BM3

Cytochrome P450s are powerful catalysts to be used for the synthesis of pharmaceuticals, plastics, and hormones. However, their industrial application is limited. One of the main bottleneck of P450s is their low coupling efficiency towards non-natural substrates. The presence of extra water around the active site is deemed to be related to uncoupling. Herein, we engineered the access tunnels of P450 BM3 from Bacillus megaterium to control water access from bulk solvent to the active site. Nine residues located in tunnels were investigated by site saturation mutagenesis to reduce the water diffusion, therefore, improving the coupling efficiency. Finally, the variant N319L/T411V/T436Q showed a 1.7-fold improved coupling efficiency and a 1.4-fold improvement of enzymatic activity towards substrate α-isophorone compared with wild type P450 BM3. Tunnel polarity analysis and MD simulation further proved that reduced water molecules around the active site could lead to higher coupling efficiency. These results demonstrated that tunnel engineering is a powerful strategy to control water diffusion and tune the coupling efficiency in P450s.

Shuaiqi Meng was supported by a Ph.D. scholarship from the China Scholarship Council (CSC No. 201906880011).

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Meng, S., Ji, Y., Liu, L., Davari, M. D., Schwaneberg, U., ChemSusChem,

  Tunnel Engineering p450 Protein Copyright: © ChemSusChem

Figure. Hydrophobic residues were introduced to P450 BM3 access tunnels, reducing extra water entering the active site. The access tunnels with increased hydrophobicity have a positive effect on the catalytic activity and coupling efficiency towards P450 BM3’s reaction.