SITE-DIRECTED MUTAGENESIS OF PUTATIVE SUBSTRATE RECOGNITION SITES IN CYTOCHROME-P450-2B11 - IMPORTANCE OF AMINO-ACID RESIDUE-114, RESIDUE-290, AND RESIDUE-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 (19 92)] were mutated to the residues found in 2B1 or 2B5. The mutants wer e expressed initially in COS cells and screened for activity toward an drostenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). P450 2B 11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and 1475S sh owed no differences from wild-type P450 2B11 in metabolite profiles wi th either substrate. Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coil for further study with androstenedione, testosterone, 7-ethoxyco umarin, (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-H CB were unaltered. For D290I, activities toward all substrates were de creased, except for 16 beta-hydroxylation of testosterone. The activit y of L363V was increased 5-6-fold for 16 alpha-hydroxylation of andros tenedione and testosterone but was decreased to 40-50% of wild-type ac tivity 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 su perimposition of the 11 mutated 2B11 residues on a P450 101 three-dime nsional model suggest that only residues 114, 290, and 363 represent s ubstrate contact residues, in excellent agreement with the experimenta l results. The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivit y of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.