title: 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.
authors.affiliation: Department of Human and Animal Biology, University of Turin, via Accademia Albertina 13, 10123, Turin, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Di Nardo
authors.forename: Andrea
authors.initials: A
authors.lastname: Fantuzzi
authors.forename: Anastasia
authors.initials: A
authors.lastname: Sideri
authors.forename: Paola
authors.initials: P
authors.lastname: Panicco
authors.forename: Carlo
authors.initials: C
authors.lastname: Sassone
authors.forename: Carlo
authors.initials: C
authors.lastname: Giunta
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009251:NADPH-Ferrihemoprotein Reductase; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases