Exploring 3-ketosteroid 9x-hydroxylases of Rhodococcus rhodochrous DSM43269

PhD ceremony: Mr. J.M. Petrusma, 14.30 uur, Aula Academiegebouw, Broerstraat 5, Groningen

Dissertation: Exploring 3-ketosteroid 9x-hydroxylases of Rhodococcus rhodochrous DSM43269

Promotor(s): prof. L. Dijkhuizen

Faculty: Mathematics and Natural Sciences

3-Ketosteroid 9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in microbial steroid degradation. The study of Mirjan Petrusma is one of the first reports on the preparation of active purified KSH enzyme. This breakthrough in the preparation of purified and active KSH enzymes subsequently allowed us to perform a more detailed characterization of the enzyme. The data strongly suggests that the five KshAs of Rhodococcus rhodochrous are involved in the degradation of different groups of steroids. Furthermore, the KshA homologues appear to play a role at different levels of the sterol catabolic pathway. Petrusma hypothesizes that the multiplicity of KshAs allows a flexible response to environmental changes, enabling a more efficient growth of the bacterium on the various steroids that may become available in nature. With the aid of a 3D structure of a KshA enzyme we mutated KshA to identify amino acids important for the functionality of KSH. This insight into KSH may result in the construction of enzymes with a better or new functionality; this is industrially relevant for the production of bioactive steroids. Furthermore Petrusma shows for the first time that KSH is active on estrogens. Thus, KSH enzymes could be used to reduce biologically harmful levels of estrogens in the environment. KSH of the notorious human pathogen M. tuberculosis is essential for its pathogenicity. The detailed insights obtained for this important enzyme provide new leads for the development of inhibitors to be used as drugs to combat tuberculosis.