Identification by in vitro mutagenesis of the interaction of two segments of C2MstC1, a chimera of cytochromes P450 2C2 and P450 2C1.
A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.
Ramarao
M K
MK
Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign 61801.
Straub
P
P
Kemper
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D006900:Hydroxylation; D007668:Kidney; D007850:Lauric Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017931:DNA Primers; D007850:Lauric Acids; D011993:Recombinant Fusion Proteins; C030358:lauric acid; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; C084930:cytochrome P-450 2C2