Molecular engineering of microsomal P450 2a-4 to a stable, water-soluble enzyme.
Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar Km value for steroid 15 alpha-hydroxylase activity (19.7 microM in comparison to 14.2 microM of the wild-type). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.
Sueyoshi
T
T
Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Park
L J
LJ
Moore
R
R
Juvonen
R O
RO
Negishi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D066298:In Vitro Techniques; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D014867:Water
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014867:Water; D003577:Cytochrome P-450 Enzyme System