Spectroscopic and kinetic evidence for redox cycling, catalase and degradation activities of MnIII(TPPS) in a basic aqueous peroxide medium

MnIII(TPPS) was found to react rapidly with hydrogen peroxide in basic aqueous solution to form intermediate (TPPS)MnV[double bond, length as m-dash]O and (TPPS)MnIV[double bond, length as m-dash]O species which, in the presence of excess H2O2, are reduced fully back to MnIII(TPPS) with clear evidence for redox cycling of MnIII(TPPS). The system shows very strong catalase and degradation activities

Enzymatic Δ1-dehydrogenation of 3-ketosteroids – Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

Data and analysis supporting the publication titled 'Enzymatic Δ1-dehydrogenation of 3-ketosteroids – Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism' (2021) created by Michał Glanowski, Patrycja Wójcik, Magdalena Procner, Tomasz Borowski, Dawid Lupa, Przemysław Mielczarek, Maria Oszajca, Katarzyna Świderek, Vicent Moliner, Andrzej J. Bojarski, Maciej Szaleniec. Δ1-Dehydrogenation of 3-ketosteroids catalyzed by FAD-dependent 3-ketosteroid dehydrogenases (Δ1-KSTD) is a crucial step in steroid degradation and synthesis of several steroid drugs. The catalytic mechanism assumes the formation of a double bond in two steps, proton abstraction by tyrosyl ion and a rate-limiting hydride transfer to FAD. This hypothesis was never verified by quantum-mechanical studies despite contradictory results from kinetic isotope effect (KIE) reported in ’60 by Jerussi and Ringold (Biochemistry 1965, 4 (10)). In this paper, we present results that reconcile the mechanistic hypothesis with experimental evidence. Quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations show that the proposed mechanism is indeed the most probable, but barriers associated with substrate activation (13.4-16.3 kcal/mol) and hydride transfer (15.5-18.0 kcal/mol) are very close (1.7-2.1 kcal/mol) which explains normal KIE values for steroids labeled either at C1 or C2 atoms. We confirm that tyrosyl ion acting as the catalytic base is indeed necessary for efficient activation of the steroid. We explain the lower value of the observed KIE (1.5-3.5) by the nature of the free energy surface, the presence of diffusion limitation and to a smaller extent conformational changes of the enzyme upon substrate binding. Finally, we confirm the Ping-Pong bi bi kinetics of the whole Δ1-dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation and enzyme reoxidation proceed at comparable rates. This repository contains data acquired in this study i.e., raw data from stopped-flow spectrophotometer used to obtain kinetic traces for steady-state and pre-steady-state kinetics, including measurements of the kinetic isotope effect. The data were fitted with kinetic models yielding kinetic constants and confirming the Ping-Pong bi bi mechanism. The pre-steady-state kinetics conducted at different micro and macroviscosites were used to measure Kinetic Solvent Viscosity Effects (KSVE). Furthermore, a pre-steady-state experiment with 17-methyltestosterone was subjected to a global-fitting procedure in Octave which resulted in establishing microkinetic constants of substrate binding and release, constant of substrate oxidation/FAD reduction as well as of the reverse process

Structure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new putative membrane-associated domain and proton-relay system in catalysis

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid 1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium substituted substrates. The role of the active site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modelling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modelling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation

Structure, mutagenesis, and QM:MM modeling of 3-ketosteroid Δ1\Delta^{1}-dehydrogenase from Sterolibacterium denitrificans : the role of a new putative membrane-associated domain and proton-relay system in catalysis

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping–Pong bi–bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation