No abstract

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-Delta4-steroids mainly in 15beta-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.

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.