Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli.
We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.
Larson
J R
JR
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
Coon
M J
MJ
Porter
T D
TD
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011817:Rabbits
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating