The self‐sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5‐hydroxylation of diclofenac

Abstract

Abbreviations: ALA, aminolevulinic acid; BM3, cytochrome P450 from Bacillus megaterium; cdw, cell dry weight; cww, cell wet weight; HPLC, high--performance liquid chromatography; IMAC, immobilised metal affinity chromatography; IPTG, isopropyl β--D--1--thiogalactopyranoside; IR, infrared; MS, mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; NMR, nuclear magnetic resonance; NSAID, non--steroidal anti--inflammatory drug; P450, cytochrome P450 monooxygenase; RhF, cytochrome P450 from Rhodococcus sp. strain NCIMB 9784. 4Abstract P450 monooxygenases are able to catalyse the highly regio--and stereoselective oxidations of many organic molecules. However, the scale--up of such bio--oxidations remains challenging due to the often--low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti--inflammatory drug that is persistent in the environment along with the 4'-- and 5--hydroxy metabolites. Here we have used the self--sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyse the highly regioselective oxidation of diclofenac to 5--hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5--hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram--scale production of human drug metabolites through recombinant whole cell biocatalysis. 5

Panel of New Thermostable CYP116B Self‐Sufficient Cytochrome P450 Monooxygenases that Catalyze C−H Activation with a Diverse Substrate Scope

“…A range of activity was observed that includes demethylation, epoxidation, hydroxylation and sulfoxidation. Previously, P450‐RhF has been shown to possess a good activity towards diclofenac ( 1 ) and produce the human metabolite 5‐hydroxydiclofenac ( 2 ) . In this study, P450‐AT, AX, JT and TT all displayed better conversions (>85 %) to the hydroxylated product than RhF (63 %) under the screening conditions.…”

“…The P450 concentration determined by CO binding was obtained using cell‐free lysates, the plots of which are shown in Figure A and include that of P450‐RhF for comparison. Each of the P450s from this study displayed a greater thermal stability than RhF, which was encouraging given the previous issues with the stability of P450‐RhF . P450 AT and JT showed modest improvements from RhF, whereas AX, TB and TT showed significant improvements with increases in T 50 of greater than 15 °C.…”

“…Residual activity experiments were conducted using purified protein (Figure B) and demonstrate not only thermal stability but also confirm the self‐sufficient nature of these P450s. RhF is not stable during routine purification and is, therefore, not included for comparison . Similar to the data shown in Figure A, the P450s that exhibit the greatest stability determined by residual activity were TB, AX and TT.…”

“…For example, the RhF reductase domain was fused to the engineered P450 Prava and utilised in the single‐step fermentative production of the cholesterol‐lowering drug pravastatin . However, the stability of both the native and artificial constructs is poor, and previous results show that upon purification the native enzyme is largely inactive because of the instability of the FMN‐FeS reductase domain …”

Strategies for the construction of insect P450 fusion enzymes

“…Furthermore, insect P450s could also be deployed as industrial enzymes for the production of fine chemicals and pharmaceuticals that cannot be produced economically by total chemical synthesis [31] or for the development of new processes based on bioelectrocatalysis [32]. Finally, they could be used for remediation purposes, such as the removal of pesticide residues and other xenobiotics from wastewater and the environment [33].…”