Y. Nonaka et al., Structure/function relationship of CYP11B1 associated with Dahl's salt-resistant rats - Expression of rat CYP11B1 and CYP11B2 in Escherichia coli, EUR J BIOCH, 258(2), 1998, pp. 869-878
Dahl's salt-resistant normotensive rats (DR rats) have been previously repo
rted to express cytochrome P-450 (CYP11B1) containing five missense mutatio
ns [Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami, H., Ogihara,
T. Be Okamoto, M. (1993) J, Biol. Chem. 268, 9117-9121]. To investigate st
ructure-function relationships of CYP11B, wild-type rat CYP11B1 and CYP11B2
and DR-CYP11B1 (mutant CYP11B1 in Dahl's salt-resistant rats) have been su
ccessfully expressed in Escherichia coil. Steroid 11 beta-hydroxylase (11 b
eta-OHase) activity observed with DR-CYP11B1 was similar to that of wildtyp
e CYP11B1, while 18-hydroxylase (18-OHase) activity of DR-CYP11B1 was lower
than that of wildtype CYP11B1. Mutant CYP11B1s containing a single or a do
uble amino acid substitution associated with DR-CYP11B1 have been also expr
essed in E. coli to investigate effects of the substitutions on enzymatic a
ctivity. Each of the single mutant enzymes showed lower 18-OHase activity t
han wild-type CYP11B1, but not as low as DR-CYP11B1. A double mutant CYP11B
1 with V381L and I384L showed 18-OHase activity at a similar low level to t
hat of DR-CYP11B1. The 19-hydroxylation (19-OHase) activity of DR-CYP11B1 w
as about one-third of that of the wild-type enzyme and,this low activity ap
peared 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 b
e responsible for the normotension of the DR rats when fed a high-salt diet
.