Steroids are ubiquitous contamination of water and soil environments and serve as a carbon and energy source for many microorganisms. Their degradation was observed in sseveral species from Actinobacteria and Proteobacteria, especially for the members of the genus Rhodococcus. Likewise, pathogenic microorganisms of eukaryotic host like Mycobacterium tuberculosis developed the ability for steroid degradation or modification. For all steroid degraders one of the key enzymes in a catabolic pathway is flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid Δ1-dehydrogenase (KstD), which catalyzes
1,2-dehydrogenation of androst-4-en-3,17-dione (AD) to androst-1,4-dien-3,17-dione (ADD).
Fig. 1. The catalytic 1,2-dehydrogenation catalyzed by KstD (AcmB) from S. denitrificans.
In our lab we study biocatalytic properties of several KstD from Sterolibacterium denitrificans Chol-1, Rhodococcus erythropolis, and Pseudomonas putida. We are interested both in fundamental studies of the reaction mechanism as well as in the application of these enzymes to synthesis of 1-dehydrosteroids. To achieve the former goal we mainly characterize these enzymes with kinetic studies (steady-state and stopped-flow kinetics, measurement of kinetic isotope effects), introduce mutations into crucial residues in the active site, study structure with spectroscopic and crystalographic techniques, as well as apply a range of theoretical methods to describe the energetics of the catalytic process (MD, QM:MM and QM:MD).
The former goal is pursued by reaction engineering, enzyme immobilization and whole-cells reactor tests, coupled with a wide screening of potential new substates that are not physiologically converted by KstD enzymes.
These studies are conducted with the financial support of two grants from National Science Center Poland: OPUS 2016/21/B/ST4/03798 "The mechanism of regioselective oxidative dehydrogenation of 3-ketosteroids catalyzed by
Δ1-cholest-4-en-3-one dehydrogenase from Sterolibacterium denitrificans" and Miniatura 2018/02/X/ST4/01963 "Searching for novel bacterial ketosteroid dehydrogenases for oxidative dehydrogenation of steroids"
Publications on the topic:
K. Sofinska, A. M. Wojtkiewicz, P. Wójcik, O. Zastawny, M. Guzik, A. Winiarska, P. Waligórski, M. Cieśla, J. Barbasz, M. Szaleniec, "Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques", Biochim. Biophys. Acta-Gen. Subj., 1863 (2019) 1027-1039
M. Tataruch, P. Wójcik, A. M. Wojtkiewicz, K. Zaczyk, K. Szymańska, M. Szaleniec, "Application of Immobilized Cholest-4-En-3-One Δ1-Dehydrogenase from Sterolibacterium Denitrificans for Dehydrogenation of Steroids", Catalyst, 10(12) (2020) 1460
A. M. Wojtkiewicz, P. Wójcik, M. Procner, M. Flejszar, M. Oszajca, M. Hochołowski, M. Tataruch, B. Mrugała, T. Janeczko, M. Szaleniec, "The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans", J. Steroid Biochem. Mol. Biol., 202 (2020) 105731
P. Wójcik, M. Glanowski, A. M. Wojtkiewicz, A. Rohman, M. Szaleniec, "Universal capability of 3‑ketosteroid Δ1‑dehydrogenases to catalyze Δ1‑dehydrogenation of C17‑substituted steroids", Microb. Cell Fact., 20 (2021) 119
M. Glanowski, P. Wójcik, M. Procner, T. Borowski, D. Lupa, P. Mielczarek, M. Oszajca, K. Świderek, V. Moliner, A.J. Bojarski, M. Szaleniec, "Enzymatic Δ1‑Dehydrogenation of 3‑Ketosteroids - Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism", ACS Catal., 11 (2021) 8211−8225
Patents and patent applications:
P. Wójcik, A. M. Wojtkiewicz, M. Tataruch, J. Morzycki, M. Szaleniec, "Sposób wytwarzania (25R)-spirosta-1,4-dien-3-onu z diosgenonu", Polish Patent Application P.433249 (13.03.2020)
A. Rugor, M. Szaleniec, T. Janeczko, M. Dymarska, E. Kostrzewa-Susłow, "Sposób wytwarzania propionianu androst-1,4-dien-3-on-17-olu", Polish Patent P.413209
A. Rugor, M. Szaleniec, T. Janeczko, M. Dymarska, E. Kostrzewa-Susłow, "Sposób wytwarzania octanu androst-1,4,6-trien-3-on-17-olu", Polish Patent P.413207
A. Rugor, M. Szaleniec, T. Janeczko, M. Dymarska, E. Kostrzewa-Susłow, "Sposób wytwarzania 17a-metyloandrost-1,4-dien-3-on-17-olu", Polish Patent P.413208