Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, ...functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.
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•Bacterial Cellulose was fully redispersed after co-drying with carboxymethyl cellulose.•The formulation was able to effectively stabilize an isohexadecane-in-water ...emulsion.•Bacterial Cellulose fibre networks structuring the emulsion were observed.•The formulation performed better than plant celluloses, and similarly to xanthan.•The bacterial cellulose formulation was able to lower the oil/water interface tension.
Bacterial cellulose (BC) is an emerging alternative to plant cellulose in different applications. Several works demonstrated the potential of never-dried BC; however, envisioning real industrial applications, a dry product retaining its functional properties upon rehydration is preferable.
A dry and completely redispersible formulation of BC with carboxymethyl cellulose (CMC) was prepared by Spray-drying. The obtained material showed a Zeta Potential of (-67.0 ± 3.9) mV, a Dv(50) of (601 ± 19.7) μm and was able to decrease the oil/water interface energy.
The dry BC:CMC formulation was employed as a stabilizer in oil-in-water emulsions, in parallel with commercial plant celluloses and Xanthan gum. The emulsions were monitored over time by optical microscopy and characterized by rheological measurements. BC:CMC effectively stabilized emulsions against coalescence and creaming, at a concentration of 0.50 % - contrarily to other commercial dry celluloses – due to the Pickering effect and to the structuring of the continuous phase, as seen with Cryo-SEM.
A covalently bound flavin cofactor is predominant in the succinate‐ubiquinone oxidoreductase (SQR; Complex II), an essential component of aerobic electron transport, and in the menaquinol‐fumarate ...oxidoreductase (QFR), the anaerobic counterpart, although it is only present in approximately 10 % of the known flavoenzymes. This work investigates the role of this 8α‐N3‐histidyl linkage between the flavin adenine dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT calculations, classical molecular‐dynamics simulations and quantum‐mechanics calculations for Complex II:FAD and Complex II:FADH2, with and without the covalent bond, were performed. It was observed that the covalent bond is essential for the active‐center arrangement of the FADH2/FAD cofactor. Removal of this bond causes a displacement of the isoalloxazine group, which influences interactions with the protein, flavin solvation, and possible proton‐transfer pathways. Specifically, for the noncovalently bound FADH2 cofactor, the N1 atom moves away from the His‐A365 and His‐A254 residues and the N5 atom moves away from the glutamine‐62A residue. Both of the histidine and glutamine residues interact with a chain of water molecules that cross the enzyme, which is most likely involved in proton transfer. Breaking this chain of water molecules could thereby compromise proton transfer across the two active sites of Complex II.
Controlling access: The role of the 8α‐N3‐histidyl linkage between the flavin adenine dinucleotide (FAD) cofactor and respiratory Complex II in the conversion between FADH2 and FAD is investigated. The presence of this covalent linkage promotes a completely different position of the isoalloxazine ring that modulates water accessibility to the FAD cofactor, thus controlling the stabilization of the cofactor and proton transfer between the two active sites of Complex II (see picture; Arg=arginine, Gln=glutamine, Glu=glutamate, His=histidine).
Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale ...underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO– groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.
Fiber meshes of poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate)/poly(ethylene oxide) (PHB/PEO) with different concentrations of chlorhexidine (CHX) were prepared by electrospinning for ...assessment as a polymer based drug delivery system. The electrospun fibers were characterized at morphological, molecular and mechanical levels. The bactericidal potential of PHB and PHB/PEO electrospun fibers, with and without CHX, was investigated against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by disk diffusion susceptibility tests. Electrospun fibers containing CHX exhibited bactericidal activity. PHB/PEO-1%CHX displayed higher CHX release levels and equivalent antibacterial activity when compared to PHB/PEO with 5 and 10 wt% CHX. Bactericidal performance of samples with 1 wt% CHX was assessed by Colony Forming Units (CFU), where reductions of 100% and 99.69% against E. coli and S. aureus were achieved, respectively.
Phenylacetone monooxygenase is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenases family, and therefore it is an ideal candidate for the synthesis of industrially ...relevant ester or lactone compounds. However, its limited substrate scope has largely limited its industrial applications. Linear substrates are interesting from an industrial point of view, it is thus necessary to identify the essential spatial requirement for achieving high conversions for non-native linear substrates. Here using molecular dynamics simulations, we compared the conversion of a non-native linear substrate 2-octanone and the native substrate phenylacetone, catalyzed by the WT enzyme and a quadruple variant P253F/G254A/R258M/L443F that exhibits significantly improved activity towards 2-octanone. We uncovered that a remarkable movement of L289 is crucial for a reshaping of the active site of the quadruple variant so as to prevent the aliphatic substrate from moving away from the C4a-peroxyflavin, thus enabling it to keep a catalytically relevant pose during the oxygenation process. By performing steady-state kinetic analysis of two single-mutation variants at position 258, we further validated that the L289 reposition is attributed to the combined effect of quadruple mutations. In order to further explore the substrate scope of PAMO we also studied the binding of cyclopentanone and 2-phenylcyclohexanone, which are the typical substrates of CPMO in group I and CHMO in group III, respectively. Our study provides fundamental atomic-level insights in rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.
Glutathione transferases are enzymes of the cellular detoxification system that metabolize a vast spectrum of xenobiotic and endobiotic toxic compounds. They are homodimers or heterodimers and each ...monomer has an active center composed of a G‐site in which glutathione (GSH) binds and an H‐site for the electrophilic substrate. When GSH binds to the G‐site, the pKa value of its thiol group drops by 2.5 units; this promotes its deprotonation and, therefore, produces a strong nucleophilic thiolate that is able to react with the electrophilic substrate. The mechanism behind the deprotonation of the thiol group is still unknown. Some studies point to the fact that the GSH glutamyl α‐carboxylate group is essential for GSH activation, whereas others indicate the importance of the active‐center water molecules. On the basis of QM/MM calculations, we propose a mechanism of GSH activation in which a water molecule, acting as a bridge, is able to assist in the transfer of the proton from the GSH thiol group to the GSH glutamyl α‐carboxylate group, after an initial GSH conformational rearrangement. We calculated the potential of mean force of this GSH structural rearrangement that would be necessary for the approach of both groups and we then performed a QM/MM ONIOM scan of water‐assisted proton transfer. The overall free‐energy barrier for the process is consistent with experimental studies of the enzyme kinetics.
Bridging the gap: It is proposed that, after an initial conformational rearrangement of glutathione (GSH), a water molecule, acting as a bridge, is able to transfer the proton from the GSH thiol group to the GSH glutamyl α‐carboxylate group (see models).
The thermal and hydrolytic degradation of electrospun gelatin membranes cross-linked with glutaraldehyde in vapor phase has been studied. In vitro degradation of gelatin membranes was evaluated in ...phosphate buffer saline solution at 37 °C. After 15 days under these conditions, a weight loss of 68% was observed, attributed to solvation and depolymerization of the main polymeric chains. Thermal degradation kinetics of the gelatin raw material and as-spun electrospun membranes showed that the electrospinning processing conditions do not influence polymer degradation. However, for cross-linked samples a decrease in the activation energy was observed, associated with the effect of glutaraldehyde cross-linking reaction in the inter- and intra-molecular hydrogen bonds of the protein. It is also shown that the electrospinning process does not affect the formation of the helical structure of gelatin chains.
Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond ...isomerization of Δ5-androstene-3,17-dione (Δ5-AD) and Δ5-pregnene-3,20-dione (Δ5-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Δ5-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Δ5-AD C4 atom to the Δ5-AD C6 atom, with a rate limiting activation energy of 15.8 kcal·mol–1. GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the O3 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leu111, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Δ4-AD crystal structure and is consistent with all available experimental data.
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