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Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA. The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase.

This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Eschenichia coli as enzymatically active fusion proteins containing the flavoprotein domain of rat NADPH-cytochrome P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by engineering the cDNAs for the steroid-metabolizing bovine adrenal P450 17A with the cDNA for rat liver NADPH-P450 reductase with the introduction of a Ser-Thr linker to 30/min in the absence of added phospholipid. Addition of purified rat liver cytochrome b5 stimulated the 17,20-lyase reaction for the conversion of 17-hydroxypregnenolone to dehydroepiandrosterone, and addition of purified rat NADPH-cytochrome P450 reductase enhanced the formation of t -1 metabolites from lauric and arachidonic acids. NADPH oxidation was tightly coupled to substrate hydroxylation with the purified fusion proteins.Cytochromes P450 (P450s) of mammalian tissues can be categorized into two groups (1) depending on their intracellular compartmentation: P450s associated with the endoplasmic reticulum are one type, and these microsomal P450s require only an NADPH-reactive, FAD-and FMNcontaining flavoprotein for the transfer of electrons from NADPH to a P450; P450s associated with mitochondria are a second type, and these mitochondrial P450s function with a mini electron-transfer chain composed of an NADPHreactive FAD-containing flavoprotein and a P450-reactive iron-sulfur protein. Many different P450s have been used to reconstitute enzymatic activities by incubating a purified P450 with its designated purified electron-transfer partner(s) in the presence of a suitable phospholipid (2).Miura and Fulco (3) were the first to describe the presence of a P450 as a fusion protein-i.e., a single protein containing both the heme domain of a P450 and the flavoprotein domain equivalent to the microsomal FAD-and FMN-containing NADPH-P450 reductase. They isolated and purified this soluble P450BM-3, named P450 102, from Bacillus megaterium and characterized it as an c-hydroxylase of fatty acids. P450 102 is the most enzymatically active form of any known P450 (turnover number > 1500/min).Murakami et al. (4) genetically engineered the cDNA of rat liver P450c (P450 lA1) with the cDNA of rat NADPH-P450 reductase to construct a P450 fusion protein which they expressed in yeast. Subsequent studies by this group (5) extended their technique to the expression in yeast of fusion proteins of P450s active in steroid metabolism by using the cDNAs of bovine P450 17A or bovine P450 21 and the cDNA of the yeast flavoprotein NADPH-P450 reductase. An enhanced enzymatic activity for the fusion protein containing bovine P450 17A in the hydroxylation of progesterone was reported (6).Studies of P450s have been greatly facilitated by the successful application of heterologous expression techniques (7). This approach has been particularly useful when applied to P450s engineered for site-specific mutations (8) or for the generation of large quantities of difficult-to-obtain bio...

Fifteen different secondary and tertiary methyl amines have been examined as substrates for the cytochromes P-450 of rat-liver microsomes to determine the similarities or differences between the NADPH and oxygen-dependent N-demethylation reaction and the reaction occurring in the presence of hydrogen peroxide. No apparent correlation of the rates of formaldehyde formation using the two different conditions of oxidation was observed. The types of cytochromes P-450 were altered by using rat-liver microsomes from animals treated with various inducing agents. No obvious predictable dependence on the animals treated with various inducing agents. No obvious predictable dependence on the type of cytochrome P-450 present was obtained for the hydrogen peroxide-supported peroxidatic reaction. It is concluded that the hydrogen peroxide-dependent N-demethylation reaction occurs by a reaction mechanism distinct from that occurring during the mixed-function oxidase activity of cytochrome P-450 obtained in the presence of NADPH and oxygen.