Expression of Cytochrome-P450-3A5 in Escherichia Coli: Effects of 5′ Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities

Abstract

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5′-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Rim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)−1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of ∼40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6β-hydroxylation, aflatoxin 3α-hydroxylation and 8,9-epoxidation, N-ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6β-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of N-ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems.