Sterolibacterium denitrificans Chol-1ST (DSMZ 13999T) is a facultative anaerobic ß-proteobacteria, closely related to Thauera and Azoarcu sp., which utilize nitrates (or oxygen in aerobic conditions) as electron acceptors. It can use cholesterol as the sole carbon source. In anaerobic conditions, the pathway starts with the oxidation of cholesterol to cholest-5-en-3-on catalyzed by cholesterol dehydrogenase/isomerise AcmA (Anaerobic cholesterol metabolism enzyme A). The third step of the pathway is the hydroxylation of C-25 tertiary carbon atom, which results in the formation of the 25-hydroxycholet-4-en-3-on. In the alternate pathway, the intermediate product, cholest-5-en-3-on, can be first oxidized to cholesta-1,4-dien-3-on by cholest-4-en-3-one-1-dehydrogenase (AcmB) and only then to the 25-hydroxy derivative (25-hydroxycholest-1,4-dien-3-on). 

S25DH reaction scheme of cholest-4-en-3-on hydroxylation to 25-hydroxycholet-4-en-3-on.

In JLBEC we have conducted both fundamental and applied studies on S25DH. As a result, we have proposed mechanistic hypothesis describing its reactivity (based on the hypothesis of EBDH) as well as developed an efficient method for regioselective hydroxylation of sterols and their derivates at C25 atom. 

List of publications on the topic:

  1. P. Kalimuthu, A. M. Wojtkiewicz, M. Szaleniec, P. V. Bernhardt, "Electrocatalytic Hydroxylation of Sterols by Steroid C25 Dehydrogenase from Sterolibacterium denitrificans", Chem. Eur. J., 24 (30) (2018) 7710-7717

  2. M. Szaleniec, A. M. Wojtkiewicz, R. Bernhardt, T. Borowski, M. Donova, "Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms", Appl. Microbiol. Biotechnol., 102(19) (2018) 8153-8171

  3. A. Rugor, M. Tataruch, J. Staroń, A. Dudzik, E. Niedzialkowska, P. Nowak, A. Hogendorf, A. Michalik-Zym, D. B. Napruszewska, A. Jarzębski, K. Szymańska, W. Białas, M. Szaleniec, "Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase", Appl. Microbiol. Biotechnol., 101 (2017) 1163-1174

  4. E. Niedzialkowska, B. Mrugala, A. Rugor, P.C. M, A. Skotnicka, J.J. Cotelesage, G.N. George, M. Szaleniec, W. Minor, K. Lewinski, "Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization", Protein Expr. Purif., 134 (2017) 47-62

  5. A. Rugor, A. Wójcik-Augustyn, E. Niedzialkowska, S. Mordalski, J. Staroń A. Bojarski, M. Szaleniec, "Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase – homology model, reactivity and isoenzymatic diversity", J. Inorg. Biochem., 173 (2017) 28–43

  6. J. Heider, M. Szaleniec, K. Sünwoldt , M. Boll, "Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation", J. Mol. Microbiol. Biotechnol., 26 (2016) 45-62


Granted patent:

  • M. Szaleniec, A. Rugor, A. Dudzik, M. Tataruch, K. Szymańska, A. Jarzębski, "Sposób otrzymywania 25-hydroksylowanych pochodnych sterolowych, w tym 25-hydroksy-7-dehydrocholesterolu", Polish Patent, P.411750-A1

Steroid C25 dehydrogenase

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