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Abstract

Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis.

S L Born, G H John, G R Harlow and J R Halpert
Drug Metabolism and Disposition July 1995, 23 (7) 702-707;
S L Born
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G H John
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G R Harlow
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J R Halpert
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Abstract

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.

 

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Drug Metabolism and Disposition
Vol. 23, Issue 7
1 Jul 1995
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Abstract

Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis.

S L Born, G H John, G R Harlow and J R Halpert
Drug Metabolism and Disposition July 1, 1995, 23 (7) 702-707;

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Abstract

Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis.

S L Born, G H John, G R Harlow and J R Halpert
Drug Metabolism and Disposition July 1, 1995, 23 (7) 702-707;
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