Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3.
The cytosolic domain of microsomal P450 52A3 (P450Cm1) was isolated as a soluble and functionally active protein. The NH2-terminal region that anchors the P450 protein to the endoplasmic reticulum was removed by sequence-specific proteolysis at a designed cleavage site. For that purpose, P450Cm1 was genetically engineered to establish at position 63-66 the sequence Ile-Glu-Gly-Arg, which is recognized by the restriction protease factor Xa. The modified P450 was produced in high yields as an integral membrane protein in Saccharomyces cerevisiae. In the microsomal fraction, it was accessible to factor Xa digestion, releasing a readily soluble, shortened P450 protein. For large scale preparation of the cytosolic domain, the modified P450Cm1 was first purified and then subjected to sequence-specific proteolysis. The highly purified delta(1-66)P450Cm1 exhibited unchanged spectral characteristics and catalyzed the hydroxylation of n-hexadecane with 85% of the activity determined for full-length wild-type P450Cm1. The method developed for the preparation of the cytosolic domain of P450Cm1 may be more generally applicable to facilitate structure-function studies on membrane-bound P450 forms.
Scheller
U
U
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Federal Republic of Germany.
Kraft
R
R
Schröder
K L
KL
Schunck
W H
WH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013057:Spectrum Analysis; D013379:Substrate Specificity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System