Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

Abstract

P450 (cytochrome P450) enzymes catalyse the mono-oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7-ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7-ethoxycoumarin, was de-ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7-ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2-hydroxylation. CYP2A5 catalysed 2- and 4-hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7-beta-hydroxy-DHEA and two P450 species produced a 2-alpha-hydroxy-DHEA. Four P450 species catalysed flavanone 2- and 4-hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high-throughput screening for drug discovery.

Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis

“…A variety of bacterial CYPs have already been expressed in E. coli (Agematu et al, 2006). Also other mammalian CYPs were expressed in E. coli and applied as whole cell biocatalysts (Uno et al, 2008).…”

Metabolism of 7‐ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants

“…All plasmids including those of the CYP2C9 variants were digested with Nde I and Sal I, and the resulting fragments were subcloned into the Nde I and Sal I sites of a pCW vector containing human NADPH‐P450 reductase cDNA (Uno et al, ), obtained as described previously. To verify that the correct inserts were ligated into the plasmids, the inserts were excised and electrophoresed.…”

Functional characterization of CYP1A9 and CYP1C1 from Anguillus japonica

“…All plasmids were digested with NdeI and SalI, and the resulting fragments were sub-cloned into the NdeI and SalI sites of a pCW vector containing human NADPH-P450 reductase cDNA (Uno et al 2008b). To verify that the correct inserts were ligated into the plasmids, the inserts were excised and their size was confirmed by electrophoretic analyses.…”