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

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.

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

“…[13,14] As an example, the 11a-hydroxylation activity could be increased up to elevenfold. [29] In summary, this concept for CYP106A2-catalyzed hydroxylation presents a simple and efficient approach towards the preparative scale synthesis of hydroxylated steroid derivatives.…”

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

“…Further studies with structurally related compounds showed the conversion of abietic acid to 12α-and 12β-hydroxyabietic acid, respectively (see Scheme 4). The existence of many mutants of CYP106A2, created by directed evolution as well as rational protein design [74,75], provides an excellent source to exploit the potential of this P450 for further biotechnological applications. Due to their diverse biological properties, the production of hydroxylated diterpene derivatives could be especially useful for the pharmaceutical industry.…”

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

“…To gain further insights into the structural basis of the regioselectivity of hydroxylation, we used a homology model recently created in our group [9] to dock abietic acid into the active site with the aid of the program FlexX. [22,23] The best docking orientations for hydroxylation in the a (yellow) and b (cyan) positions ( Figure 3) revealed possible hydrogen bonds between the acid group of the substrate and the side chains of the residues D295, E78, and R66.…”

“…[9] Molecular docking calculations were performed for 580 www.chembiochem.org the substrate abietic acid. For the calculations, the FlexX docking program was used.…”

“…It hydroxylates 3-oxo-D 4 -steroids such as testosterone and progesterone mainly in the b orientation at the 15-position. [8,9,10] Additionally, the production of 6b-, [11] 9a-, and 11a-hydroxyprogesterone has been reported. [12] These hydroxylation products or their derivatives are known as pharmaceuticals or useful precursors in the synthesis of anti-inflammatory, diuretic, anabolic, contraceptive, antiandrogenic, progestational, and antitumor compounds.…”