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 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 .