Structure, Mutagenesis, and QM:MM Modeling of 3-Ketosteroid Δ1-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.

Protective Effects of Cannabidiol (CBD) against Qxidative Stress, but Not Excitotoxic-Related Neuronal Cell Damage-An In Vitro Study.

Cannabidiol (CBD) appears to possess some neuroprotective properties, but experimental data are still inconsistent. Therefore, this in vitro study aimed to compare the effects of CBD in a wide range of concentrations on oxidative stress and excitotoxic-related cell damage. Results showed that low concentrations of CBD ameliorated the H2O2-evoked cell damage of primary cortical neuronal cell culture. However, higher concentrations of CBD alone (5-25 µM) decreased the viability of cortical neurons in a concentration-dependent manner and aggravated the toxic effects of hydrogen peroxide (H2O2). Neuroprotection mediated by CBD in primary neurons against H2O2 was not associated with a direct influence on ROS production nor inhibition of caspase-3, but we found protective effects of CBD at the level of mitochondrial membrane potential and DNA fragmentation. However, CBD had no protective effect on the glutamate-induced cell damage of cortical neurons, and in higher concentrations, it enhanced the toxic effects of this cell-damaging factor. Likewise, CBD, depending on its concentration, at least did not affect or even enhance cortical cellular damage exposed to oxygen-glucose deprivation (OGD). Finally, we showed that CBD in submicromolar or low micromolar concentrations significantly protected human neuronal-like SH-SY5Y cells against H2O2- and 6-hydroxydopamine (6-OHDA)-induced cell damage. Our data indicate that CBD has a dual effect on oxidative stress-induced neuronal death-in low concentrations, it is neuroprotective, but in higher ones, it may display neurotoxic activity. On the other hand, in excitotoxic-related models, CBD was ineffective or enhanced cell damage. Our data support the notion that the neuroprotective effects of CBD strongly depend on its concentration and experimental model of neuronal death.

The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans.

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Δ1-dehydrogenation of a wide range of 3-ketosteroids. In this study, we demonstrate the application of AcmB in the synthesis of 1-dehydro-3-ketosteroids and investigate the influence of reaction conditions on the catalytic performance of the enzyme. The recombinant AcmB expressed in E. coli BL21(DE3)Magic exhibits a broad pH optimum and pH stability in the range of 6.5 to 9.0. The activity-based pH optimum of AcmB reaction depends on the type of electron acceptor (2,6-dichloroindophenol - DCPIP, phenazine methosulfate - PMS or potassium hexacyanoferrate - K3[Fe(CN)6]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (kcat/Km = 1.4·105 s-1·M-1 at pH 9.0) followed by DCPIP (kcat/Km = 1.0·105 s-1·M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5·102 s-1·M-1 at pH = 8.0). The unique feature of AcmB is its capability to convert both testosterone derivatives (C20-C22) as well as steroids substituted at C17 (C27-C30) such as cholest-4-en-3-one or (25R)-spirost-4-en-3-one (diosgenone). Apparent steady-state kinetic parameters were determined for both groups of AcmB substrates. In a batch reactor synthesis, the solubility of water-insoluble steroids was facilitated by the addition of a solubilizer, 2-hydroxypropyl-ß-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 µM of isolated enzyme, PMS and aerobic atmosphere resulting in >99% conversion of the C17-C20 3-ketosteroids within 2 h. Finally, the whole cell E. coli system with recombinant enzyme was demonstrated as an efficient biocatalyst in the synthesis of 1-dehydro-3-ketosteroids.