EXPRESSION OF CYTOCHROME-P450-3A5 IN ESCHERICHIA-COLI - EFFECTS OF 5'MODIFICATION, PURIFICATION, SPECTRAL CHARACTERIZATION, RECONSTITUTIONCONDITIONS, AND CATALYTIC ACTIVITIES

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid seque nce identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus o f a P450 3A5 cDNA was modified for optimal expression in Escherichia c oli using the vector pCW, by aligning the MALLLAVFL N-terminal sequenc e 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 3 A5 cDNA. Two constructs were made, differing by their identity with th e 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 recomb inant P450 3A4 up to codon 15, the 3A5 sequence being introduced there after. 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)]. T he recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed prev iously for P450 3A4, with an overall yield of similar to 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydr oxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, N-ethy lmorphine N-demethylation, erythromycin N-demethylation, and d-benzphe tamine N-demethylation. The reconstitution of nifedipine oxidation, te stosterone 6 beta-hydroxylation, and the aflatoxin oxidation activitie s showed dependence upon the presence of cytochrome b(5), divalent cat ions, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demeth ylations of N-ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly d ependent upon modifications of reconstitution systems. (C) 1995 Academ ic Press, Inc.