Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp EB104

CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxy lation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation an d the interaction of hydrophobic substrates with soluble enzymes. Here the molecular cloning of the CYP153 gene is reported. Single specific primer PC R was applied to yield the whole gene sequence via chromosomal walks. CYP15 3 consists of 497 amino acids (M-r = 56 kDa) and thus represents an unusual ly long bacterial P450, containing all P450 typical structural elements. It constitutes the new P450 family CYP153. The prolonged N-terminus of about 90 amino acids does not contain a so far known membrane-anchoring sequence but a 28-amino acid long amphipathic helix. The relevance of the remarkably long N-terminus and of other sequence motives like the hydrophobic F-G loo p is discussed with respect to substrate binding and recognition. (C) 2001 Academic Press.