Site-directed mutagenesis of putative substrate recognition sites in cytochrome P450 2B11: importance of amino acid residues 114, 290, and 363 for substrate specificity.
Eleven amino acid residues unique to dog cytochrome P450 (P450) 2B11, compared with rat 2B1 and 2B2, rabbit 2B4 and 2B5, and mouse 2B10, in the putative substrate recognition sites [J. Biol. Chem. 267:83-90 (1992)] were mutated to the residues found in 2B1 or 2B5. The mutants were expressed initially in COS cells and screened for activity toward androstenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). P450 2B11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and I475S showed no differences from wild-type P450 2B11 in metabolite profiles with either substrate. Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coli for further study with androstenedione, testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 245-HCB. With V114I, hydroxylation of steroids and warfarin and 2-hydroxylation of 245-HCB were decreased, whereas 7-ethoxycoumarin O-dealkylation and 3-hydroxylation of 245-HCB were unaltered. For D290I, activities toward all substrates were decreased, except for 16 beta-hydroxylation of testosterone. The activity of L363V was increased 5-6-fold for 16 alpha-hydroxylation of androstenedione and testosterone but was decreased to 40-50% of wild-type activity with 7-ethoxycoumarin and warfarin and to 6-8% of control for 2-hydroxylation of 245-HCB. Alignment of P450 2B11 with P450 101 and super-imposition of the 11 mutated 2B11 residues on a P450 101 three-dimensional model suggest that only residues 114, 290, and 363 represent substrate contact residues, in excellent agreement with the experimental results. The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivity of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.
Hasler
J A
JA
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Harlow
G R
GR
Szklarz
G D
GD
John
G H
GH
Kedzie
K M
KM
Burnett
V L
VL
He
Y A
YA
Kaminsky
L S
LS
Halpert
J R
JR
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D004285:Dogs; D004926:Escherichia coli; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D039181:Steroid 16-alpha-Hydroxylase; D013379:Substrate Specificity
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; C450239:CYP2C3 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase