New Uncharged 2-Thienostilbene Oximes as Reactivators of Organophosphate-Inhibited Cholinesterases

The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) by organophosphates (OPs) as nerve agents and pesticides compromises normal cholinergic nerve signal transduction in the peripheral and central nervous systems (CNS) leading to cholinergic crisis. The treatment comprises an antimuscarinic drug and an oxime reactivator of the inhibited enzyme. Oximes in use have quaternary nitrogens, and therefore poorly cross the brain–blood barrier. In this work, we synthesized novel uncharged thienostilbene oximes by the Wittig reaction, converted to aldehydes by Vilsmeier formylation, and transformed to the corresponding uncharged oximes in very high yields. Eight trans,anti- and trans,syn-isomers of oximes were tested as reactivators of nerve-agent-inhibited AChE and BChE. Four derivatives reactivated cyclosarin-inhibited BChE up to 70% in two hours of reactivation, and docking studies confirmed their productive interactions with the active site of cyclosarin-inhibited BChE. Based on the moderate binding affinity of both AChE and BChE for all selected oximes, and in silico evaluated ADME properties regarding lipophilicity and CNS activity, these compounds present a new class of oximes with the potential for further development of CNS-active therapeutics in OP poisoning.

The beryllium fluoride form of the Na+,K+-ATPase and the intermediates of the functional cycle

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane. Here, we describe a 4.0 Å resolution crystal structure of the pig kidney Na+,K+-ATPase stabilized by beryllium fluoride (denoted E2-BeFx). The structure shows high resemblance to the E2P phosphoenzyme obtained by phosphorylation from inorganic phosphate (Pi) and stabilised by cardiotonic steroids, and reveals a Mg2+ bound near the ion binding site II. Anomalous Fourier analysis of the crystals soaked in Rb+ (K+ congener) followed by a low resolution rigid-body refinement (6.9-7.5 Å) revealed pre-occlusion transitions leading to activation of the desphosphorylation reaction. Mg2+ location indicates a site of an initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Despite the overall structural resemblance to the Pi-induced E2P phosphoform, BeFx inhibited enzyme is able to binds both ADP/ATP and ions, the features that relate E2-BeFx complex to an intermediate of the functional cycle of the Na+,K+-ATPase prior E2P.

Degradation of epigallocatechin and epicatechin gallates by a novel tannase TanHcw from Herbaspirillum camelliae

Background Herbaspirillum camelliae is a gram-negative endophyte isolated from the tea plant. Both strains WT00C and WT00F were found to hydrolyze epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) to release gallic acid (GA) and display tannase activity. However, no tannase gene was annotated in the genome of H. camelliae WT00C. Results The 39 kDa protein, annotated as the prolyl oligopeptidase in the NCBI database, was finally identified as a novel tannase. Its gene was cloned, and the enzyme was expressed in E. coli and purified to homogeneity. Moreover, enzymatic characterizations of this novel tannase named TanHcw were studied. TanHcw was a secretary enzyme with a Sec/SPI signal peptide of 48 amino acids at the N-terminus, and it catalyzed the degradation of tannin, methyl gallate (MG), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG). The optimal temperature and pH of TanHcw activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na+, K+ Mn2+ and Triton-X100, Tween80 increased the enzyme activity of TanHcw, whereas Zn2+, Mg2+, Hg2+, EMSO, EDTA and β-mercaptoethanol inhibited enzyme activity. Km, kcat and kcat /Km of TanHcw were 0.30 mM, 37.84 s−1, 130.67 mM−1 s−1 for EGCG, 0.33 mM, 34.59 s−1, 105.01 mM−1 s−1 for ECG and 0.82 mM, 14.64 s−1, 18.17 mM−1 s−1 for MG, respectively. Conclusion A novel tannase TanHcw from H. camelliae has been identified and characterized. The biological properties of TanHcw suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase TanHcw in this study gives us a reasonable explanation for the host specificity of H. camelliae. In addition, studying the characteristics of this enzyme offers the possibility of further defining its potential in industrial application.

Influence of saccharide modifications on heparin lyase III substrate specificities

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types, and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6-O sulfation, and 3-O sulfation on lyase III digestion was examined systematically. It was demonstrated that lyase III from both sources can completely digest oligosaccharides lacking O-sulfates. 2-O Sulfation completely blocked cleavage at the corresponding site; 6-O and 3-O sulfation on glucosamine residues inhibited enzyme activity. We also observed that there are differences in substrate specificities between the two lyase III enzymes for highly sulfated oligosaccharides. These findings will facilitate obtaining and analyzing the functional sulfated domains from large HS polymer, to better understand their structure/function relationships in biological processes.

Development of MTH1-Binding Nucleotide Analogs Based on 7,8-Dihalogenated 7-Deaza-dG Derivatives

MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.

Alpha-glucosidase inhibitory activity of some brown seaweeds collected in Nha Trang bay, Khanh Hoa province

Diabetes has become a global problem in recent years. Inhibition of α-glucosidase is one of the effective approaches to control the postprandial blood glucose and thereby managing diabetes. This study evaluated inhibitory activity of seven brown seaweed extracts (Colpomenia sinuosa, Padina australis, Sargassum aquifolium, Sargassum mcclurei, Sargassum duplicatum, Sargassum polycystum and Sargassum swartzi) against α-glucosidase. The results indicated that all seaweed extracts inhibited enzyme activity with the IC50 values ranging from 154.27 to 426.27 μg/mL. The seaweed Sargassum mcclurei showed the highest α-glucosidase inhibitory activity. The effects of extraction conditions and extraction solvent fractions on α-glucosidase inhibitory activity of Sargassum mcclurei were investigated. The suitable extraction conditions were found to be the solid to liquid ratio (g/mL) of 1/40, the extraction time of 60 min and the extraction temperature of 60oC. The ethyl acetate extracted fraction showed the highest α-glucosidase inhibitory activity compared with other fractions.

Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

The Growing Complexity of UHRF1-Mediated Maintenance DNA Methylation

Mammalian DNMT1 is mainly responsible for maintenance DNA methylation that is critical in maintaining stem cell pluripotency and controlling lineage specification during early embryonic development. A number of studies have demonstrated that DNMT1 is an auto-inhibited enzyme and its enzymatic activity is allosterically regulated by a number of interacting partners. UHRF1 has previously been reported to regulate DNMT1 in multiple ways, including control of substrate specificity and the proper genome targeting. In this review, we discuss the recent advances in our understanding of the regulation of DNMT1 enzymatic activity by UHRF1 and highlight a number of unresolved questions.