Subcellular localization, aggregation state, and catalytic activity of microsomal P450 cytochromes modified in the NH2-terminal region and expressed in Escherichia coli.
This laboratory previously expressed cDNAs encoding rabbit liver cytochrome P450 2E1 (the ethanol-inducible isoform) and the corresponding protein lacking amino acids 3-29, a proposed membrane anchor, in Escherichia coli. Unexpectedly, the shortened protein, like the full-length form, was found to be predominantly located in the bacterial inner membrane rather than the cytosol and to have full catalytic activity. Additional proteins with alterations in the NH2-terminal region of P450 2E1 or P450 2B4 (the phenobarbital-inducible isoform) were similarly expressed, and it was concluded that such modifications can change the cytochrome to an increased cytosolic localization and that the first two hydrophobic segments are not uniquely involved in attachment to the bacterial membrane (Pernecky et al., 1993, Proc. Natl. Acad. Sci. USA 90, 2651-2655). In the present study, three chimeric cytochromes were produced to determine the effect on subcellular localization: 2E1:2B4, in which the first 17 residues of 2E1 (delta 3-29) replaced the corresponding 17 residues in 2B4 (delta 2-27), and BM-3:2B4 and BM-3:2E1, in which the first 19 residues of P450BM-3 replaced the first 17 in 2B4 (delta 2-27) and 2E1 (delta 3-29), respectively. Of the total cytochrome expressed, the localization in the E. coli cytosol was about 60, 70, and 80% for the respective chimeras, with 80% being the highest for any P450 we have examined. A plot of the extent of membrane binding versus hydropathy of the NH2-terminal region showed that the terminal sequence strongly influences the subcellular distribution and that a group of 2E1 proteins and a group of 2B4 proteins each have other regions that characteristically determine the extent of membrane attachment. The role of the NH2-terminal region in the high level of aggregation of purified full-length P450 is indicated by the finding that the multimeric state of 2E1 or 2B4 is unaffected by sodium cholate at concentrations that convert 2E1 (delta 3-29) or 2B4 (delta 2-27) to the monomeric state. In contrast to our earlier experience with P450 2E1, purified P450 2B4 (delta 2-27) has on the average only about half the activity of full-length 2B4 with substrates that undergo oxidative dealkylation or oxygenation at a hydroxyl group.
Pernecky
S J
SJ
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606, USA.
Olken
N M
NM
Bestervelt
L L
LL
Coon
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D010089:Oxidoreductases, N-Demethylating; D005982:Glutathione Transferase