TY - JOUR T1 - Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis. JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 702 LP - 707 VL - 23 IS - 7 AU - S L Born AU - G H John AU - G R Harlow AU - J R Halpert Y1 - 1995/07/01 UR - http://dmd.aspetjournals.org/content/23/7/702.abstract N2 - Canine hepatic cytochrome P450 PBD-2 metabolizes 2,2',4,4',5,5'-hexachlorobiphenyl and catalyzes the 21-hydroxylation of progesterone, thereby distinguishing PBD-2 as unique among 2B P450s. Heterologous expression of the PBD-2 cDNA, P450 2B11, in COS and yeast systems produced a protein capable of androstenedione metabolism; however, this P450 did not metabolize progesterone in a manner consistent with PBD-2. Modification of PBD-2 reconstitution parameters resulted in significantly increased catalytic activities and further emphasized differences between PBD-2 and the heterologously expressed enzyme. Subsequent Escherichia coli expression of 2B11 generated a protein that possessed substrate specificities indistinguishable from those of PBD-2 and provided a system in which the determinants of 2B11 progesterone 21-hydroxylation could be examined via site-directed mutagenesis. Site-directed mutants of 2B11 expressed in E. coli revealed that substitution of Ile with Val at position 363 converted 2B11 into a highly active and specific progesterone 16 alpha-hydroxylase. Mutants Val-114 --> Ile, Asp-290 --> Ile, and Ile-365 --> Phe exhibited decreased progesterone 21- and 16 alpha-hydroxylase activities, in accordance with decreases in androstenedione hydroxylase activities. In contrast, replacement of Ile-365 with Val or Leu resulted in much greater changes in progesterone than androstenedione hydroxylation. Thus, the combination of P450 reconstitution techniques, heterologous expression, and site-directed mutagenesis has revealed PBD-2 to be an important progesterone 21-hydroxylase in canine liver and has identified several amino acid residues that alter progesterone metabolism by 2B11. ER -