Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the blood pressure and has been reported to play a key role in the formation of arterial hypertension, heart failure and myocardial fibrosis. Aldosterone synthase (CYP11B2) catalyzes the biosynthesis of aldosterone by successive 11β- and 18-hydroxylation followed by an 18-oxidation of 11-deoxycorticosterone and thus comprises an important drug target. For more than 20 years, all attempts to purify recombinant human CYP11B2 in significant amounts for detailed analysis failed due to its hydrophobic nature as a membrane protein. Here, we present the successful expression of the protein in E. coli yielding approx. 90 nmol/l culture, its purification and detailed enzymatic characterization. Biochemical analyses have been performed using in vitro conversion assays which revelead a V(max) of 238±8 nmol products/nmol hCYP11B2/min and a K(m) of 103±8 μM 11-deoxycorticosterone. Furthermore, binding analyses indicated a very loose binding of the first intermediate of the reaction, corticosterone with a K(d) value of 115±6 μM whereas for 11-deoxycorticosterone a K(d) of 1.34±0.13 μM was estimated. Upon substrate conversion of 11-deoxycorticosterone, new intermediates have been identified as 19- and 18-hydroxylated products not described before for the human enzyme. To understand the differences in substrate conversion, we constructed a new homology model based on the 3D structure of CYP11A1, performed docking studies and calculated the activation energy for hydrogen abstraction of the different ligands. The data demonstrated that the 11β-hydroxylation requires much less abstraction energy than hydroxylation at C18 and C19. However, the C18 and C19 hydroxylated products might be of clinical importance. Finally, purified CYP11B2 represents a suitable tool for the investigation of potential inhibitors of this protein for the development of novel drugs against hypertension and heart failure as was shown using ketoconazole.
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