Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

Abstract

When the cDNA encoding bovine microsomal 17a-hydroxylase cytochrome P450 (P45017a) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017a per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017a can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017a enzyme. Surprisingly, it is found that E. coli contain an electrontransport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17a-hydroxylase and 17,20-lyase activities of P45017a. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structurefunction analysis of the cytochrome P450 system. Microsomal cytochromes P450 are integral membrane hemoproteins that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous compounds. Deriving reducing equivalents from NADPH via a membrane-bound flavoprotein oxidoreductase (NADPH-cytochrome P450 reductase), these mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water. Recent study of the molecular aspects underlying eukaryotic cytochrome P450 structure and function has relied on the techniques of molecular biology to synthesize specific individual forms ofcytochrome P450 in heterologous expression systems. Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3, 4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies. This microsomal cytochrome P450 catalyzes the regiospecific and stereospecific 17a-hydroxylation of the C21 steroids pregnenolone and progesterone in the pathway leading to the production of cortisol and the 17,20-lyase conversion of 17a-hydroxypregnenolone to the C19 adrenal androgen dehydroepiandrosterone (DHEA) in the adrenal cortex of most mammalian species. P45017a also converts these 17a-hydroxylat...

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

“…The first published was a fusion of rat CYP1A1 and yeast RED 22 and was expressed in yeast. Since then several groups have constructed fusion proteins including human CYP3A4 fused to rat RED 21 and expressed them in bacteria 24,25,41 for functional studies.…”

Cinnamate 4‐hydroxylase from Catharanthus roseus and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli

“…We detected little or no protein in strain M15 which is recommended for pQE-6 derivatives, or in the strains DH5cz and JM109 which have been used successfully for the expression of animal P450 alone or as PasdP450-RED fusions (see [11,13] for some examples). Those experiments revealed unexplained differences between DHScz and JM 109 as well, and we have no simple explanation for the finding that the C4H/ P,50-RED expression was successful in DS410, but not in the others.…”

“…One of the reasons may be that these bacteria lack P450 activities [1] and thus also the characteristic cytochrome P4s0 reductase (P45o-RED) that is necessary for P45o function. Results with some animal P450 suggested that it may be replaced by the combined action of two soluble E. coli flavoproteins [10,11], but a high efficiency required purified proteins [12], and the general applicability was not investigated. The obtaining of active P450 then requires the reconstitution with purified plant P450 reductase, but that is technically tedious and difficult.…”

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

“…However, taking the lead from numerous groups that have focused on E. coli gene expression of mammalian and plant P450 enzymes (Barnes 1996;Barnes et al 1991;Chang et al 2007;Leonard et al 2006;Pritchard et al 2006;Yun et al 2006), functional expression of the first Taxol pathway P450 was accomplished (Ajikumar et al 2010). Doing so required truncation of the native transmembrane region (with three different truncation lengths tested) and further modification of the N-terminal portion of the enzyme to include an eight amino acid membrane anchor peptide sequence (MALLLAVF) which has proven functional in E. coli (Barnes et al 1991). Furthermore, as in the case of the taxadiene synthase, the P450 gene was synthesized to eliminate any codon bias (this same step was also taken for the accompanying P450 reductase), and reduced temperature induction conditions and other process parameters were modified so as to maximize eventual production (Table 1).…”

“…This can be mitigated by the native capabilities to complement reductase activity when using S. cerevisiae, but the same level of convenience may not be provided with E. coli (Barnes et al 1991;Jennewein et al 2005;Oeda et al 1985). Again thanks to work by Rodney Croteau's group, a reductase has been isolated from Taxus cuspidata (Jennewein et al 2005).…”