Expression of cytochrome P450 3A7 in Escherichia coli: effects of 5' modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase.
Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).
Gillam
E M
EM
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia. gillam@plpk.uq.edu.au
Wunsch
R M
RM
Ueng
Y F
YF
Shimada
T
T
Reilly
P E
PE
Kamataki
T
T
Guengerich
F P
FP
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis / Q000235:genetics; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013379:Substrate Specificity
D001059:Apoproteins; D002273:Carcinogens; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase