"The mechanism of regioselective oxidative dehydrogenation of 3-ketosteroids catalyzed by Δ1-cholest-4-en-3-one dehydrogenase from Sterolibacterium denitrificans"

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Prof. Maciej Szaleniec

Project Leader

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Magdalena Procner, PhD

Scientist with PhD

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Michał Glanowski, MSc

PhD student

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Olga Zastawny, MSc

PhD student

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Agnieszka M. Wojtkiewicz, PhD

Scientist with PhD

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Patrycja Wójcik, MSc

 PhD student

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Beata Mrugała, MSc

PhD student

Cultivation of bacteria, expression and isolation of the enzyme, preparation and expression of enzyme mutants

The first part of the project employs the development of the enzyme overexpression system in E. coli, its cultivation,  and optimization of enzyme purification protocol as well as  mutation of several key residues in the active site and isolation of such mutated enzymes. 

bioreactor

Theoretical and experimental
investigation of reaction mechanism

The third phase of the project will encompass theoretical studies of the possible reaction pathways. Initially, we will use homology model and the known structure of KstD from  Rodoccocus erythropolis and then use the models obtained from XRD study of the enzyme. We will use MD, QM-only, QMMM and QM:MD techniques to verify mechanistic hypotesis and to simulate kinetic isotope effect. 

qmmm model of KSTD

Acnowlegments

PROJECT OPUS 11 "The mechanism of regioselective oxidative dehydrogenation of3-ketosteroids catalyzed by Δ1-cholest-4-en-3-one dehydrogenase from Sterolibacterium denitrificans" is sponsored by the National Science Centre Poland 

Logo of National Science Center Poland

Enzyme crystalization and
determination of its 3D structure.
Study of the quaternary structure of
the enzyme

AcmB quaternary and 3D structure is up to date unknown. To determine the functional quaternary structure of the enzyme we will employ in the project a combination of biophysical techniques, such as size exclusion chromatography, A-PAGE electrophoresis, dynamic light scattering and AFM microscopy. Furthermore, we will screen for the best crystallization conditions and try to crystalize the enzyme with steroid in the active site and solve its structure with XRD.  

XRD of protein

Verification of mechanistic hypothesis by means of isotope methods, kinetics and genetic mutations of the enzyme

In parallel to theoretical modeling the AcmB catalytic properties will be characterized with kinetic methods. Steady-state kinetics will be used to establish substrate affinities and maximum reaction velocities, while the stopped-flow technique will be used to determine the kinetic rate of FAD reduction and oxidation. The experiments with the deuterated substrate will deliver experimental values of kinetic isotope effect that will be compared to theoretical predictions. 

spectra of FAD reduction by deuterated 3-ketosteroid