Role of the hydrophobic amino-terminal segment of alcohol -inducible cytochrome P-450 studied by heterologous expression.

Abstract

Much remains to be learned about the structure and function of cytochrome P-450, a versatile catalyst in detoxification and lipid metabolism, including the role of the hydrophobic amino-terminal segment. In the present study, a cDNA encoding rabbit liver cytochrome P-450 2E1, the ethanol-inducible P-450, was expressed in Escherichia coli. The expressed enzyme is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The expressed cytochrome exhibits a reduced-CO difference spectrum characteristic of P-450 2E1, and when the solubilized membranes or partially purified P-450 preparations are reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine, the enzyme has full catalytic activity. A modified 2E1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for P-450, was also expressed in E. coli, and the shortened protein was also predominantly located in the bacterial inner membrane rather than in the cytosol. When solubilized membranes were subjected to chromatography on S Sepharose, hydroxyapatite, and DEAE Sepharose, an electrophoretically homogeneous cyctochrome was obtained. The shortened P-450 has spectral characteristics identical to those of the full-length protein and the rates of catalysis with four substrates, aniline, ethanol, p-nitrophenol, and N-nitrosodiethylamine, in the reconstituted enzyme system are the same as with the full-length P-450. The apparent Km values of p-nitrophenol, the reductase, and cytochrome $b\sb5$ are unchanged as is the value for the V$\sb{\rm max}$ for the hydroxylation reaction. Furthermore, the requirement for phosphatidylcholine for full catalytic activity is not different despite the absence of the NH$\sb2$-terminal segment. Although bacterial membranes may have somewhat different properties from those of eukaryotic organisms, the results obtained indicate that our experimental approach will be useful to others interested in the complexities of cytochrome P-450 membrane topology.