Importance of successive prolines in the carboxy-terminal region of P450 2C2 and P450 2C14 for the hydroxylase activities.
Proline-480 and proline-481 are highly conserved in the P450 2 family. When these successive prolines of P450 2C2 were replaced by Ala-Thr, its activities of laurate (omega-1)-hydroxylation and benzphetamine N-demethylation were lost. On the other hand, the mutated P450 which retained one of the proline residues was 60-100% active in the (omega-1)-hydroxylation and 100-120% active in the N-demethylation. Pro-Pro (480, 481) to Ala-Thr mutated P450 2C14 was also inactive in testosterone 16 alpha-hydroxylation and benzphetamine N-demethylation, the activities of the wild-type P450 2C14. In the carboxyterminal region of the P450 2C subfamily, the sequence of P450 2C2 from residues 469-473 is noticeably different from those of the other members of this subfamily. The Ser-473 to Val mutation of P450 2C2 caused a decrease in the (omega-1)-hydroxylase activity to one-fifth but the N-demethylase activity was not much affected. When the sequence of P450 2C2 from residues 469-473 was replaced by that of P450 2C14 (mutation at four residues), the 16 alpha-hydroxylase and N-demethylase activities were increased by seven- and three-fold, respectively, and the (omega-1)-hydroxylase activity was decreased to one-third. The mutated P450 2C2, in which the carboxy-terminal four residue sequence or the proline cluster adjacent to the amino-terminal membrane-anchor signal sequences was deleted, was not accumulated in yeast cells transformed with plasmids directing the synthesis of such P450s.(ABSTRACT TRUNCATED AT 250 WORDS)
Uno
T
T
Institute for Protein Research, Osaka University.
Imai
Y
Y
Nakamura
M
M
Okamoto
N
N
Fukuda
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004795:Enzyme Stability; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry*; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry; D039181:Steroid 16-alpha-Hydroxylase; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D011392:Proline; D039181:Steroid 16-alpha-Hydroxylase