Differential effects of mutations in substrate recognition site 1 of cytochrome P450 2C2 on lauric acid and progesterone hydroxylation.
Mutations at amino acid positions 107-120, which are part of a predicted substrate recognition site [Gotoh, O. (1992) J. Biol. Chem. 267, 83-90], were analyzed in C2MstC1, a chimera of P450 2C2 and P450 2C1. This hybrid protein has a new activity for progesterone C21-hydroxylation in addition to the lauric acid (omega-1)hydroxylase activity present in both parent proteins. Various substitutions for highly conserved glycines at positions 111 and 117 and tryptophan at position 120 strongly decreased the lauric acid hydroxylase activity of P450 2C2 and C2MstC1 and the progesterone hydroxylase activity of C2MstC1. Activities of mutant proteins with substitutions at 107, 108, and 112-115 were also strongly reduced. Modest or no decreases in activity were observed for substitutions at 109, 110, 116, 118, and 119. Lauric acid hydroxylase activity decreased more in most C2MstC1 mutants than in those of P450 2C2, particularly at positions 107 and 108. A substitution of phenylalanine for valine-112 reduced progesterone hydroxylation by 30-fold while only moderately reducing lauric acid hydroxylase by 40%. This differential effect on two dissimilar substrates demonstrates the importance of residue 112 for substrate interactions. The results are consistent with a model in which residues 107-110 align with the B'-helix of the bacterial proteins P450cam and P450BM-3. This helix is followed by a substrate-contacting loop from 111 to 116, and residues 117-120 align with the C-helices of the bacterial proteins In this alignment, Trp-120 is positioned behind the heme such that it could participate in electron transfer from reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
Straub
P
P
Department of Physiology and Biophysics, University of Illinois at Urbana, Champaign 61801.
Lloyd
M
M
Johnson
E F
EF
Kemper
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006900:Hydroxylation; D007850:Lauric Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011374:Progesterone / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D007850:Lauric Acids; D011993:Recombinant Fusion Proteins; C030358:lauric acid; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C084930:cytochrome P-450 2C2; D013255:Steroid 21-Hydroxylase; D042926:Cytochrome P-450 CYP4A