Modulating the Coupling Efficiency of P450 BM3 by Controlling Water Diffusion through Access Tunnel Engineering

ChemSusChem, vol. 15, pp. e202102434

Abstract

Cytochromes P450 have gained much interest for their broad substrate scope in the catalysis of oxidation reactions for pharmaceuticals, plastics, and hormones. However, achieving high coupling efficiency by the engineering of P450s is still a big challenge. The presence of extra water around the active site is deemed to be related to uncoupling. In this study, the access tunnels of P450 BM3 from Bacillus megaterium are engineered to control water access from bulk solvent to the active site. Nine residues located in tunnels are investigated by site-saturation mutagenesis to reduce water diffusion, thereby improving the coupling efficiency. The recombined variant N319L/T411V/T436A shows improved coupling efficiency (from 31.2 % to 52.6 %). Tunnel polarity analysis and molecular dynamics simulation further indicate that reduced water molecules around the active site lead to higher coupling efficiency. Overall, this study provides valuable insight on improving coupling efficiency by controlling water diffusion through tunnel engineering.

Authors 5

1. Shuaiqi Meng Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

   These authors contributed equally to this work

2. Yu Ji Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

   These authors contributed equally to this work

3. Luo Liu

   Beijing University of Chemical Technology

   Affiliation as printed

   Beijing Bioprocess Key Laboratory Beijing University of Chemical Technology Beisanhuan East Road 15 Beijing 10029 P. R. China

   Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beisanhuan East Road 15, Beijing, 10029 P. R. China

4. Mehdi D. Davari

   Leibniz Institute of Plant Biochemistry

   Affiliation as printed

   Department of Bioorganic Chemistry Leibniz Institute of Plant Biochemistry Weinberg 3 06120 Halle Germany

   Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany

5. Ulrich Schwaneberg corresponding Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI-Leibniz Institute for Interactive Materials Forckenbeckstraße 50 52074 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany