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The three-dimensional structure of a truncated mutant of bovine adrenodoxin has been resolved at 1.85 A resolution by MAD. The protein consists of a large core region and a more flexible hairpin loop bearing residues which have been previously described as being involved in redox partner recognition. To study the role of distinct protein domains and amino acids of adrenodoxin in interaction with adrenodoxin reductase (AdR), CYP11A1 and CYP11B1, as well as in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis and produced in Escherichia coli, and their structural and functional properties have been characterized in detail. It could be demonstrated that Tyr82 is located at the edge of the flexible interaction loop of adrenodoxin participating in interactions with AdR and P450s. His56, being close to Tyr82, forms a bridge between the core region of adrenodoxin and the interaction loop. Its role in transmitting changes of the cluster region to the interaction site has also been supported by functional studies. Pro108 of adrenodoxin, the only proline residue contained in the protein and being conserved in this position among several other vertebrate-type ferredoxins, has been demonstrated to be of importance for the correct folding of this protein.

We expressed a guinea pig 11 beta-hydroxylase cDNA (1) in COS-1 cells. In order to find the optimal expression system we compared three expression plasmids, each driven by a different promoter. Although promoters exhibited different transcriptional activities this did not result in different enzymatic activities. Upon cotransfection with bovine adrenodoxin a 5-fold increase of enzyme activity was achieved. A comparison with the bovine 11 beta-hydroxylase clearly demonstrated that the guinea pig enzyme was not able to produce significant amounts of 18-hydroxylated and 18-oxidized products from deoxycorticosterone under the experimental conditions used.

The cDNAs of human 11 beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) were cloned from surgically removed normal human adrenal using polymerase chain reaction. The cloned cDNAs were transfected into COS-7 cells and steroid hydroxylase activity of the two cytochromes P450 expressed was determined in order to elucidate their the functional characteristics. Stable expression of human CYP11B1 or CYP11B2 was performed in V79 hamster cells and confirmed by Southern blotting, Northern blotting, and enzymatic activity. Interestingly, recombinant V79 cells were able to support CYP11B1 and CYP11B2 dependent steroid conversion without additional heterologous expression of the corresponding electron donor system.

We recently observed that treatment of CYP11B2-expressing COS-1 cells with the broad range kinase inhibitor, staurosporine (STS), strongly inhibited aldosterone biosynthesis, indicating that the activity of a kinase might be a prerequisite for steroid hydroxylase activity. In an attempt to identify such kinases, we measured conversion of 11-deoxycortisol (RSS) and 11-deoxycorticosterone (DOC) by V79MZh11B1 and V79MZh11B2 cells, respectively, in the presence of STS and also after treatment with the kinase inhibitors chelerythrine, rottlerin and Gö 6976. The conversion of both substrates by both cell lines was affected in a selective manner by the kinase inhibitors, suggesting that the activity of the novel PKC-delta and either of conventional PKCs or of PKD alter steroid hydroxylation activity, with their influence depending on both the cytochrome P450 tested and on its steroid substrate.

When a peptide of the 316-398th amino acid residues in CYP11B1 was inserted to the corresponding site in CYP11B2, the 18-hydroxylation activity of the chimera decreased to a level similar to that of CYP11B2. Site-directed mutagenesis studies indicated that this alteration in the activity was due to the change of one amino acid, Ser321–>Pro, which is present at the C-terminal side of enzyme's I-helix. Conversely, when the corresponding region of CYP11B2 was changed to that of CYP11B1, the 18-hydroxylation activity increased to the similar level as that of CYP11B1. This elevation of the activity seemed to be due to the exchange of two amino acid residues; one, the 321st residue and the other, the 381st located in the K-helix. The results are discussed in comparison with those obtained by the human isozymes.

CYP11B1 was detected in the human adrenal cortex and in human adenomas by in situ-hybridization methods. Specific riboprobes were generated and hybridized to sections of an Aldosterone Producing Adenoma (APA), the non-tumour portion of the corresponding adrenal gland and two adenomas not related to hyperaldosteronism. P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis. Semi-quantitative analysis has been performed and seems to be applicable for a further classification of adrenal tumours. Stable expression of CYP11B1 cDNA was performed in V79 cells. The interference of different substances (metyrapone, spironolactone and different imidazole derivatives) with CYP11B1 activity was studied using this cell line. The cell line revealed to be suitable for analysis of the active site of CYP11B1 as well as for analysis of side effects of drugs on steroidogenesis.

Mutants of the adrenal ferredoxin (adrenodoxin) have been expressed in E. coli in order to improve the understanding of its structure and function. Replacement of the ligands to the /2Fe-2S/ center, C46, C52, C55 and C92, by serine, histidine or aspartic acid lead to apoproteins not incorporating the iron-sulfur cluster, whereas C95S forms a functionally active holoprotein. C-terminal deletions up to amino acid 109 affect the conformation around the iron-sulfur cluster in adrenodoxin and the interaction with CYP11A1 and CYP11B1, but not with adrenodoxin reductase. The presence of P108 is necessary for incorporation of the /2Fe-2S/ cluster and obviously for correct folding of adrenodoxin.