Structure/function relationship of CYP11B1 associated with Dahl's salt-resistant rats--expression of rat CYP11B1 and CYP11B2 in Escherichia coli.
Dahl's salt-resistant normotensive rats (DR rats) have been previously reported to express cytochrome P-450 (CYP11B1) containing five missense mutations [Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami H., Ogihara, T. & Okamoto, M. (1993) J. Biol. Chem. 268, 9117-9121]. To investigate structure-function relationships of CYP11B, wild-type rat CYP11B1 and CYP11B2 and DR-CYP11B1 (mutant CYP11B1 in Dahl's salt-resistant rats) have been successfully expressed in Escherichia coli. Steroid 11beta-hydroxylase (11beta-OHase) activity observed with DR-CYP11B1 was similar to that of wild-type CYP11B1, while 18-hydroxylase (18-OHase) activity of DR-CYP11B1 was lower than that of wild-type CYP11B1. Mutant CYP11B1s containing a single or a double amino acid substitution associated with DR-CYP11B1 have been also expressed in E. coli to investigate effects of the substitutions on enzymatic activity. Each of the single mutant enzymes showed lower 18-OHase activity than wild-type CYP11B1, but not as low as DR-CYP11B1. A double mutant CYP11B1 with V381L and I384L showed 18-OHase activity at a similar low level to that of DR-CYP11B1. The 19-hydroxylation (19-OHase) activity of DR-CYP11B1 was about one-third of that of the wild-type enzyme and this low activity appeared due to the V443M mutation. These results suggest that three of five amino acid substitutions present in DR-CYP11B1 account for the decreased 18-OHase and 19-OHase activities. A decrease in these enzyme activities may be responsible for the normotension of the DR rats when fed a high-salt diet.
Nonaka
Y
Y
Department of Basic Laboratory Sciences, Osaka University Medical School, Japan. ynonaka@sahs.med.osaka-u.ac.jp
Fujii
T
T
Kagawa
N
N
Waterman
M R
MR
Takemori
H
H
Okamoto
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D003900:Desoxycorticosterone / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D020303:Rats, Inbred Dahl; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism; D013256:Steroids / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D013256:Steroids; D003900:Desoxycorticosterone; D013250:Steroid Hydroxylases; C027110:steroid 19-hydroxylase; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase