The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function.

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b5. The cDNAs of b5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b5 proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b5. Purified b5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b5 for the rapid transfer of electrons from NADPH.

Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase.

A fusion protein containing the heme domain of bovine cytochrome P450 17A and the flavin domains of rat NADPH-cytochrome P450 reductase has been genetically engineered by linking the modified cDNAs for each gene with the codons for serine and threonine. Transformation of Escherichia coli (DH5 alpha) and growth under defined conditions permits expression of 600-700 nmol of membrane-bound fusion protein per liter of growth medium (approximately 4% of cellular protein). A method has been developed for the solubilization, isolation, and purification to homogeneity of this protein. In the presence of NADPH the purified fusion protein catalyzes the 17 alpha-hydroxylation of progesterone and pregnenolone as well as the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone. The 17,20-lyase activity is enhanced sixfold by the addition of purified rat liver cytochrome b5. Further, dehydroepiandrosterone is slowly metabolized to a number of additional more polar metabolites while 17 alpha-hydroxy-progesterone is slowly converted to dihydroxy-progesterone metabolites as well as a small amount of androstenedione in a reaction not influenced by cytochrome b5. Use of 5 alpha-pregnan steroids as substrates show the importance of the 3 beta-hydroxyl group for cytochrome b5 stimulated 17,20-lyase activity. Studies investigating the factors affecting electron transport between the flavin and heme domains suggest that the protein exists as a tight complex functioning as a self-contained biocatalytic unit.

High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.

This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Escherichia 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 give a protein we have named rF450[mBov17A/mRatOR]L1. Similarly, the cDNA for the omega-hydroxylase of rat liver (P450 4A1) was linked with the cDNA for rat liver NADPH-P450 reductase to give rF450[mRat4A1/mRatOR]L1. A procedure involving disruption of transformed E. coli by sonication, isolation of membranes by differential centrifugation, solubilization with detergent, and affinity chromatography provided significant amounts of purified fusion proteins of approximately 118 kDa. The purified fusion proteins had turnover numbers for the metabolism of steroids (rF450[mBov17A/mRatOR]L1) or fatty acids (rF450[mRat4A1/mRatOR]L1) ranging from 10/min 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 omega--1 metabolites from lauric and arachidonic acids. NADPH oxidation was tightly coupled to substrate hydroxylation with the purified fusion proteins.

High-level expression of functional human cytochrome P450 1A2 in Escherichia coli.

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.

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator.

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)

Some enzymatic vagaries of a bovine adrenal microsomal cytochrome P-450 introduced and expressed in transformed monkey kidney cells.

The present studies illustrate the ability to carry out kinetic measurements of steroid hydroxylation using a cytochrome P-450 expressed in a tissue culture cell system. For these experiments a single species of cDNA, incorporated into a suitable expression vector, has been introduced via transfection. A number of interesting preliminary observations have been made on the function of the cytochrome P-450 associated with adrenocortical microsomes which catalyses the 17-hydroxylation of progesterone and pregnenolone. In confirmation of earlier reports the adrenal 17-OHase possesses both 17-hydroxylase as well as C17,20-lyase activities. However, the latter is only functional with 17-OH pregnenolone and not with 17-OH progesterone as substrate. This result differs from the numerous reports that a lyase activity for both substrates is associated with this P-450. The reason for this difference between a delta 4 and a delta 5 steroid remains unresolved although initial experiments indicate that the 5-alpha reduced progesterone is a suitable substrate for both the 17-OHase as well as lyase reactions. This result suggests an inhibitory effect of the delta 4 double bond preventing the carbon-carbon cleavage of the C-17,20 bond in 17-OH progesterone. Clearly more experiments will be required to resolve this question. Measurements of substrate affinity for the cytochrome P-450 expressed in COS cells appears to be influenced by a permeability barrier to the steroid effecting the transport of the steroid across the cell membrane into the cells. This conclusion is suggested by the presence of a time lag before the onset of metabolism as well as by the discrepancy in the concentration of substrate required to give half-maximal rates of metabolism, cf. the results obtained where the initial concentration of progesterone present in the reaction medium is altered versus those experiments measuring the kinetics of substrate depletion. The presence of such a barrier to the free movement of steroid across the membrane is interesting to contemplate when considering the build up of 17-OH pregnenolone required for the lyase reaction. Most unexpected where the results obtained when a comparable expression vector containing the cDNA for cytochrome b5 was cotransfected with pCD17 alpha 2. Cytochrome b5 has been postulated to be an electron transfer component participating in the cyclic function of some cytochromes P-450 (Hildebrandt and Estabrook, 1971).(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of a fibric acid type of hypolipidemic agent on the oxidative metabolism of arachidonic acid by liver microsomal cytochrome P-450.

The regiospecificity of arachidonic acid oxygenation, catalyzed by rat liver microsomal fractions in the presence of NADPH, can be altered by animal pretreatment with a fibric acid type of hypolipidemic drug, ciprofibrate. While microsomal fractions isolated from either control or phenobarbital-treated animals oxygenate arachidonic acid to mainly epoxyeicosatrienoic acids (EETs), animal pretreatment with ciprofibrate results in an eightfold stimulation of omega and omega-1 oxidation, concomitant with a net decrease in the formation of both HETEs and EETs. The isomeric composition of the EETs and of the omega and omega-1 oxidation products formed is also dependent on the type of animal pretreatment. Associated decreases in the amounts of HETEs and the rate of hydrogen peroxide formation suggests a modification of the "uncoupler action" of arachidonic acid during the function of different cytochromes P-450.

The peroxidatic function of liver microsomal cytochrome P-450: comparison of hydrogen peroxide and NADPH-catalysed N-demethylation reactions.

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.

The reaction of arachidonic acid epoxides (epoxyeicosatrienoic acids) with a cytosolic epoxide hydrolase.

Epoxyeicosatrienoic acids, formed during the cytochrome P-450-catalyzed oxidation of arachidonic acid, react with a liver cytosolic epoxide hydrolase to form vicinal diols of eicosatrienoic acid. The role of this cytosolic enzyme, rather than a microsomal bound type, explains previous results illustrating the ability to accumulate epoxides during the in vitro aerobic steady state of oxidative metabolism of arachidonic acid by liver microsomes. The inability of the 5,6-epoxyeicosatrienoic acid to serve as a suitable substrate for this enzyme is discussed in light of recent studies concerning possible unique physiological functions for this metabolite.