Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes

Abstract

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119-131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography-mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The KM values calculated for these compounds were in the millimolar range: 1.21+/-0.07 mM for chlorzoxazone, 2.52 +/- 0.08 mM for aniline, 0.81+/-0.04 mM for propranolol. The values of vmax for chlorzoxazone and propranolol were 46.0+/-9.0 and 7.6+/-3.4 nmol min-1 nmol-1, respectively. These values are higher then those measured for the human enzymes. The vmax value for aniline was 9.4+/-1.3 nmol min-1 nmol-1, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

“…The direct use of human cytochrome P450 is limited by the need of a redox partner and the poor stability and activity. However, bacterial counterparts such as cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, can be directly used [3] or engineered to metabolise drugs and to produce the same metabolites as the human enzymes [4][5][6][7][8][9][10][11][12][13][14]. This protein is a selfsufficient fatty acids monoxygenase containing a NADPH-dependent reductase (BMR) and a P450 catalytic domain (BMP) fused in a single polypeptidic chain [15,16].…”

A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite Synthesis

“…The wildtype CYP102A1 enzyme, moreover, is able to metabolise certain xenobiotics including chlorzoxazone. [21] The introduction of specific mutations was reported to additionally broaden the substrate specificity of CYP102A1. Especially the single mutation F87V proved to be important.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…On the other hand, bacterial P450 enzymes are known to be well coupled and as in the case of P450 BM3 from Bacillus megaterium, have been shown to be able to produce drug metabolites typical of the human enzymes with a high coupling efficiency (Di Nardo et al, 2007;Di Nardo and Gilardi, 2012). This has led to a wide range of protein engineering studies on this enzyme to widen its catalytic abilities (Tsotsou et al, 2012(Tsotsou et al, , 2013Ryu et al, 2014;Di Nardo et al, 2015;Ren et al, 2015;Capoferri et al, 2016).…”