Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s.
A proline-rich region is present following the signal-anchor sequence in the amino-terminal portion of all known microsomal cytochrome P-450s. To assess the functional significance of the proline residues in this region, we systematically altered these residues of cytochrome P450(M1) (P450 2C11); one, two, and three proline residues out of the five in the region were exchanged for alanine residues. The wild-type and the mutated proteins were expressed in the fission yeast Schizosaccharomyces pombe under the control of nmt1 promoter. The wild-type and the mutated proteins were all highly expressed in the yeast cells (5-9% of the total membrane protein). The expressed wild-type P450(M1) showed a typical carbon monoxide difference spectrum of P-450 and the activity of testosterone hydroxylation, whereas all the mutated proteins constructed in the present study showed no characteristic P-450 spectrum, suggesting that the substitution of the proline residues in this region resulted in a defect of proper heme incorporation. Furthermore, the mutated proteins in which more than one proline residues had been exchanged were more sensitive to trypsin digestion than the wild type. From these results, we propose that the proline residues in the proline-rich region are crucial for the formation of the correct conformation of microsomal P-450 molecules.
Yamazaki
S
S
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Sato
K
K
Suhara
K
K
Sakaguchi
M
M
Mihara
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D011392:Proline; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase