Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis.
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.
Born
S L
SL
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
John
G H
GH
Harlow
G R
GR
Halpert
J R
JR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004285:Dogs; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D000882:Haplorhini; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011374:Progesterone / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D039181:Steroid 16-alpha-Hydroxylase; D013255:Steroid 21-Hydroxylase / Q000378:metabolism*
D007527:Isoenzymes; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D039181:Steroid 16-alpha-Hydroxylase; D013255:Steroid 21-Hydroxylase