Expression of human liver cytochrome P450 IIIA4 in yeast

Abstract

Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage (pVNF25) showed a ferrous -CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIEl did not recognize yeastexpressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1 ,Coxidation, quinidine 3-hydroxylation and Noxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH -cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPHcytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.Cytochromes P-450 (P450s) are members of a superfamily of heme proteins that catalyze the oxidation of a wide variety of endogenous and exogenous compounds including most drugs and carcinogens, the existence of multiple enzymes being the origin of the broad substrate specificity (Gonzalez, 1989). This enzyme multiplicity is now well established on the basis of purification, amino acid composition, electrophoretic and

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

“…CYP2B6, 2C8, 2C9 and 3A4 were expressed in a previously described yeast strain W(R)fur1 [36] system, in which yeast cytochrome P450 reductase was overexpressed. Transformation by a pYeDP60 vector containing one of the human liver CYP2B6, 2C8, 2C9 and 3A4 cDNAs was then performed according to a general method of construction of yeast strain W(R)fur 1 expressing various human liver P450s [37][38][39]. Yeast culture and microsomes preparation were performed by using previously described techniques [40].…”

Gene structure of CYP3A4, an adult‐specific form of cytochrome P450 in human livers, and its transcriptional control

“…The catalytic properties of CYP3 A4 have been examined extensively using the expressed enzyme. This enzyme catalyzes the oxidation of various steroids [9, 14, 151 and xenobiotics [8,9,[14][15][16][17][18]. The same enzyme is also capable of activating various promutagens to mutagens 19, 14, 17, 20-221. Comparing the reported sequences, we found that the identity between CYP3A4 and CYP3A7 was 95% between nucleotides and 87% between amino acids.…”

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

“…The application of heterologous expression system to study various aspects of individual enzyme such as elucidation of structure-function relationships and discernment of catalytic specificity has been widely used in recent years. For these reasons, different recombinant systems such as yeast (Renaud et al 1990), bacteria (Gilliam et al 1993) and insects (Zhang et al 2004) have been developed to understand how to influence on co-operativity exhibited by cytochrome P450.…”