Recombinant human cytochrome P450 1A2 and an N-terminal-truncated form: construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPH-cytochrome P450 reductase.
Previous work from this laboratory indicated that the N-terminus of recombinant human cytochrome P450 (P450) 1A2 expressed in Escherichia coli is blocked (P. Sandhu, Z. Guo, T. Baba, M. V. Martin, R. H. Tukey, and F. P. Guengerich, (1994) Arch. Biochem. Biophys. 30, 168 -177). A modification of this construct was done to insert an extra 12 residues containing a thrombin-sensitive site just beyond the most N-terminal hydrophobic segment, and the protein was expressed, purified, and cut with thrombin. Treatment of E. coli membranes in which the P450 1A2 with 12 extra residues was present with thrombin did not release the truncated form, suggesting that the added thrombin site may be imbedded in the membrane. The N-terminal of the recombinant proteins were blocked but mild acid hydrolysis generated the expected Met residues as analyzed by Edman degradation. Laser light scattering studies indicated that purified thrombin-cleaved P450 1A2 (devoid of the usual N-terminal 25 residues or the first 36 residues of the wild-type protein) was still aggregated in the absence of detergent and that some nondenaturing detergents could reduce the apparent size to that of a tetramer. The N-terminal truncated protein was as catalytically active as full-length P450 1A2 but required a higher concentration of NADPH-P450 reductase. P450 1A2 exhibited catalytic activity in E. coli cells, and activity of the purified enzyme could be supported by E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase could also substitute for NADPH-P450 reductase. These artificial electron donors did not require phospholipid for oxidation reactions; however, phospholipid was required for optimal activity when either P450 1A2 or the truncated form was used with NADPH-P450 reductase. Rates of oxidation of 7-ethoxyresorufin were considerably higher for both P450 1A2 and the truncated form when NADPH-P450 reductase was replaced with the "oxygen surrogate" iodosylbenzene, indicating that P450 reduction and oxygen activation are normally limiting in this P450 1A2 reaction.
Dong
M S
MS
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Yamazaki
H
H
Guo
Z
Z
Guengerich
F P
FP
D000595:Amino Acid Sequence; D001483:Base Sequence; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005288:Ferredoxins / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion
D005288:Ferredoxins; D005418:Flavodoxin; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase