Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.

Abstract

Human cytochrome P450 3A4is recognized as the catalyst for the oxygen-dependent metaboism of a diverse group of medicafly important chemicals, inWuding the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids, such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymaticafly active fusion protein (rF450[mHum3A4/mRatOR]Ll) has been expressed at high levels in Escherichia coli and purified to homogeneity.Enzymatic studies show a requirement for lipid, detergent, and cytochrome bs for the 63-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometricafly detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolim of both testosterone and erthromycin. These resuits relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.One ofthe most versatile ofthe cytochrome P450s is the form present in human liver called CYP3A4, which catalyzes the oxidative metabolism of a wide array of different chemicals with markedly different structural characteristics (1). Interest has centered on this P450 since it is reported to be one of the more abundant P450s in human liver (2); it is inducible by a variety of agents including glucocorticoids as well as phenobarbital (3); it appears to play a central role in the metabolism of the immunosuppressive cyclic peptide cyclosporin A as well as macrolide antibiotics, such as erythromycin (4); it also catalyzes the 63-hydroxylation of a number of steroids including testosterone, progesterone, and cortisol (5). Clinical interest relates to the measurement of erythromycin metabolism by a breath test (6) and the presence of6f-hydroxylated steroids in urine (7) as indicators of CYP3A4 function for evaluation of transplant recipients.P450s ofthe 3A family were first characterized by Guzelian and colleagues (8) based on the ability ofthe catabolic steroid pregnenolone-16a-carbonitrile to induce a unique form of P450 (which they called P-450p). They also recognized the ability of the macrolide antibiotic triacetyloleandomycin (TAO) to serve as a powerful inducer of this type of P450-a result of interest because of the known ability of TAO and other compounds to form stable, metabolite-inhibitor complexes with P450 (9). Reconstitution studies using purified P450s of the 3A family have shown the need to include phospholipids, detergents, and cytochrome b5 when testing enzymatic activities (10, 11).

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.…”

“…Co-expression can be achieved either as two separate proteins 19 or with the construction of a fusion protein linking CYP2B6 and RED in one gene. Previous efforts to fuse the two genes have resulted in active proteins; [21][22][23][24][25] however, fusion proteins have never been expressed in mammalian cells and RED has never been used in association with CYP2B6. Finally, the notion of fusing two human proteins should not produce a host immune response.…”

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

“…Anionic lipids, particularly PtdSer, favour the formation of the ultimate mutagen DE2 to the detriment of that of the far less carcinogenic DE1. It has been reported that lipids affect several catalytic activities of CYP3A4 [40,41]. Thus, it would be interesting to know whether other enzymatic activities of human CYP1A1 also depend on the membrane structure and/or lipids.…”

Attachment of the N-Terminal Domain of Salmonella typhimurium AhpF to Escherichia coli Thioredoxin Reductase Confers AhpC Reductase Activity but Does Not Affect Thioredoxin Reductase Activity

“…Rates were adjusted by subtracting the very low rate of NAD(P)H consumption by the flavoproteins in the absence of AhpC. Kinetic data were fit to the Michaelis-Menten equation (39)(40)(41) and the ferredoxin/ ferredoxin-NADP + reductase (Fd/FNR) fusion protein (42). In each of these cases, design of the fusion proteins was guided by naturally occurring fusions of related proteins.…”