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

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.

Flexibility Regulation of Loops Surrounding the Tunnel Entrance in Cytochrome P450 Enhanced Substrate Access Substantially

“…The catalytic efficiency ( k cat / K m ) of all nine variants was higher than that of CYP116B3 WT (12.5–134-fold, Table ), and especially the best variant 8G8 (A86T/T91L/M108N/A109M/T111A) showed a significant 134-fold improvement. Besides, there was a positive correlation of activity and coupling efficiency, and the latter represents the utilization efficiency of NADPH . The nine best variants with high dealkylation activity also exhibited improved coupling efficiency (33–48%) compared with CYP116B3 WT (9%) (Table ).…”

“…Besides, there was a positive correlation of activity and coupling efficiency, and the latter represents the utilization efficiency of NADPH. 33 The nine best variants with high dealkylation activity also exhibited improved coupling efficiency (33−48%) compared with CYP116B3 WT (9%) (Table 1). Investigation of Static Tunnel Properties of CYP116B3 WT and Variants.…”

“…Among the 2085 clones with dealkylation activity toward 7-ethoxycoumarin, 339 clones with a two-fold greater dealkylation activity than CYP116B3 WT were rescreened. The screening procedures were as follows: single colonies from the CYP116B3 SSM library were transferred into 96-deep-well microtiter plates (MTPs; 2.2 mL polypropylene plates; Brand GmbH, Wertheim, Germany) for cultivation and expression . Each well, containing 600 μL of TB medium supplemented with 50 mg/L kanamycin, 5 mg/L thiamine, and 0.1 mM IPTG, was inoculated with a single colony.…”

“…The screening procedures were as follows: single colonies from the CYP116B3 SSM library were transferred into 96-deep-well microtiter plates (MTPs; 2.2 mL polypropylene plates; Brand GmbH, Wertheim, Germany) for cultivation and expression. 33 Each well, containing 600 μL of TB medium supplemented with 50 mg/L kanamycin, 5 mg/ L thiamine, and 0.1 mM IPTG, was inoculated with a single colony. Cells were grown at 18 °C and 900 rpm for about 22 h. The plates were centrifuged at 4000 rpm and 4 °C for 30 min, and the supernatants were discarded.…”

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

“…One key factor may be the hydrophobic substrate tunnel, which allows for poor accessibility of H 2 O 2 to the heme center (buried deep inside of the P450s) during catalysis. Although H 2 O 2 may also reach the heme center via water tunnels connecting the active site to the bulk solvent environment, the requirements in terms of the number of water molecules in the active site during catalysis may imply specific gating mechanisms for the water tunnel, which would be unfavorable for a continuous supply of H 2 O 2 . Given quite similar physicochemical features of H 2 O 2 with water, e.g., almost the same molecular diameter (0.25–0.28 nm [H 2 O 2 ] vs ca.…”