Michael Lisurek

Modulation of aldosterone and cortisol synthesis on the molecular level.

CYP11B1 and the closely related CYP11B2 are involved in the production of adrenal steroid hormones. Although in human their primary structure is 93% identical they are involved in the biosynthesis of functionally diverse products, such as glucocorticoids and mineralocorticoids, respectively. In contrast, bovine CYP11B1 combines both activities in one single enzyme. The CYP11B family belongs to class I cytochromes P450 that have been described in bacteria and mitochondria and receive their electrons from a low molecular weight iron sulphur protein which is reduced by a NADPH-dependent FAD-containing reductase. In this review, we summarise the current knowledge on the modulation of aldosterone and cortisol synthesis by transcriptional regulation, on the molecular level as consequence of mutations found in patients suffering from steroid hormone-related diseases as well as introduced by site-directed mutagenesis and as consequence of protein-protein interaction with both CYP11A1 and the natural redox partner adrenodoxin.

The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation.

Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono- and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3Dstructure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-α-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly- but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors.

Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2

The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept

Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive compounds. Furthermore, secondary assay-validated hits presented in this study show the practical relevance of our library design strategy.

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3‐oxo‐Δ4‐steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15β‐position. We combined a high‐throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the Vmax and Km values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12α‐ and 12β‐hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole‐cell catalyst for the selective allylic 12α‐ and 12β‐hydroxylation was applied to produce the hydroxylated products.

Function and engineering of the 15β-hydroxylase CYP106A2

CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

Theoretical and Experimental Evaluation of a CYP106A2 Low Homology Model and Production of Mutants with Changed Activity and Selectivity of Hydroxylation

Steroids are important pharmaceutically active compounds. In contrast to the liver drug‐metabolising cytochrome P450s, which metabolise a variety of substrates, steroid hydroxylases generally display a rather narrow substrate specificity. It is therefore a challenging goal to change their regio‐ and stereoselectivity. CYP106A2 is one of only a few bacterial steroid hydroxylases and hydroxylates 3‐oxo‐Δ4‐steroids mainly in 15β‐position. In order to gain insights into the structure and function of this enzyme, whose crystal structure is unknown, a homology model has been created. The substrate progesterone was then docked into the active site to predict which residues might affect substrate binding. The model was substantiated by using a combination of theoretical and experimental investigations. First, numerous computational structure evaluation tools assessed the plausibility of its protein geometry and its quality. Second, the model explains many key properties of common cytochrome P450s. Third, two sets of mutants have been heterologously expressed, and the influence of the mutations on the catalytic activity towards deoxycorticosterone and progesterone has been studied experimentally: the first set comprises six mutations located in the structurally variable regions of this enzyme that are very difficult to predict by cytochrome P450 modelling (K27R, I86T, E90V, I71T, D185G and I215T). For these positions, no participation in the active‐site formation was predicted, or could be experimentally demonstrated. The second set comprises five mutants in substrate recognition site 6 (S394I, A395L, T396R, G397P and Q398S). For these residues, participation in active‐site formation and an influence on substrate binding was predicted by docking. These mutants are based on an alignment with human CYP11B1, and in fact most of these mutants altered the active‐site structure and the hydroxylation activity of CYP106A2 dramatically.

Deletions in the loop surrounding the iron–sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450scc (CYP11A1)

The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.

Cyanobacterial electron carrier proteins as electron donors to CYP106A2 from Bacillus megaterium ATCC 13368

The CYP450 from Bacillus megaterium (BmCYP106A2) catalyzes the 15beta-hydroxylation of several steroids and also synthesizes mono-hydroxylated 9alpha- and 11alpha-OH-progesterone. This study reports on the ability of BmCYP106A2 to be efficiently reduced by the photosynthetic flavodoxin and, particularly, ferredoxin electron carriers from the cyanobacterium Anabaena. These results open the possibility for the design of a hybrid system to provide reducing equivalents for the hydroxylation process. Additionally, they suggest that despite the interaction of BmCYP106A2 with these proteins, particularly with flavodoxin, they do not rely on a precise complementarity of the reacting molecules, rearrangements might be required and alternative binding modes might contribute to the observed electron transfer reactions.

Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α‐Hydroxylated Products from C‐19 Steroids

Cytochromes P450 catalyze a variety of synthetically useful reactions. However, it is difficult to determine their physiological or artificial functions when a plethora of orphan P450 systems are present in a genome. CYP260A1 from Sorangium cellulosum So ce56 is a new member among the 21 available P450s in the strain. To identify putative substrates for CYP260A1 we used high‐throughput screening of a compound library (ca. 17 000 ligands). Structural analogues of the type I hits were searched for biotechnologically relevant compounds, and this led us to select C‐19 steroids as potential substrates. We identified efficient surrogate redox partners for CYP260A1, and an Escherichia coli‐based whole‐cell biocatalyst system was developed to convert testosterone, androstenedione, and their derivatives methyltestosterone and 11‐oxoandrostenedione. A detailed 1H and 13C NMR characterization of the product(s) from C‐19 steroids revealed that CYP260A1 is the very first 1α‐steroid hydroxylase.