Mechanism Of Cleavage Research Articles

Mechanistic Insights into the Anaerobic Degradation of Globally Abundant Dihydroxypropanesulfonate Catalyzed by the DHPS-Sulfolyase (HpsG).

2(S)-Dihydroxypropanesulfonate (DHPS) is the main abundant organosulfonate in the biosphere generated by the microbial degradation of the abundant organosulfur species 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose, SQ). Massive amounts of DHPS can also be produced by the highly abundant oceanic diatoms. The quantity of degradation DHPS is so large that it has become an important part of the earth's sulfur. The recently characterized O2-sensitive glycyl radical enzyme DHPS-sulfolyase HpsG in anaerobic bacteria was found to be capable of cleaving the C-S bond of DHPS under anaerobic conditions. However, the detailed degradation mechanism is still unclear. Here, on the basis of the crystal structure of HpsG, we constructed the computational model and performed QM/MM calculations to illuminate the anaerobic degradation mechanism of DHPS. Our calculations revealed that the degradation reaction follows an unusual radical-dependent mechanism that does not require a conserved Glu464 to deprotonate the C2 hydroxyl of substrate to promote the C-S cleavage; instead, after the first hydrogen abstraction triggered by the thiyl radical (Cys462), the C-S bond in 2(S)-dihydroxypropanesulfonate can directly collapse. Thus, conserved Glu464 mainly plays a role in stabilizing the substrate and reaction intermediate by forming a hydrogen bond. After the release of the sulfonic acid group from the protein environment, the deprotonated Glu464 spontaneously accepts a proton from the C2 hydroxyl of the substrate radical. Our findings clarified an unusual C-S cleavage mechanism involved in the DHPS degradation reaction catalyzed by GREs.

A new method for the toughness assessment of mobile crane components based on damage mechanics

The characterization of toughness properties in standard Charpy or fracture mechanics tests calls for thickness requirements to be met. Therefore, the characterization of toughness properties is a problem for thin-walled structures. Replacing Charpy impact toughness testing by impact notch tensile testing can solve this problem. However, the toughness requirements are still expressed in terms of standard test results. Therefore, a framework is proposed here for translating these standard test requirements into impact notch tensile test requirements. The proposed framework relies on numerical simulations with a phenomenological damage mechanics model, which uses state-of-stress-dependent, strain-based criteria for the prediction of local damage and global fracture. This model takes the effects of non-proportional strain paths into account and applies different criteria for cleavage and ductile fracture in order to predict correctly the activation of cleavage and ductile fracture mechanisms in the corresponding numerical simulations.

Modulating levels of cell surface CD6 is a novel mechanism for regulating T cell activity.

Abstract CD6 is a co-stimulatory receptor expressed on the surface of T cells that promotes immune synapse formation, T cell activation, and migration into tissues by engaging its ligand, ALCAM. CD6high cells have been described to be more pathogenic relative to CD6low cells. Furthermore, T cells in CD6 knockout mice have decreased proliferation and inflammatory responses. Hence, a therapeutic that can modulate surface levels of CD6 may have favorable outcomes for autoimmune/inflammatory conditions. We recently demonstrated that the anti-CD6 mAb, itolizumab, induces proteolytic cleavage of cell surface CD6. The goal of the work shown here is to characterize the mechanism of cleavage and the downstream functional consequences of reduced cell surface CD6. Treatment of PBMCs with itolizumab induces CD6 cleavage from the cell surface and a concomitant increase in the soluble form detected in the supernatant. Itolizumab-induced loss of CD6 was not observed with isolated T cells, however CD6 levels were reduced by 87% when T cells and monocytes were cocultured with itolizumab. The cleavage of CD6 is initiated through rapid cell-to-cell contact between T cells and monocytes via FcγRI. The dose-dependent reduction in surface levels of CD6 positively correlates with decreases in T cell activation markers such as CD25, PD-1, and CD71 (Pearson, p<0.001) and cytokine production including IL-2 (p<0.01) and TNFα (p<0.05). Itolizumab-treated cells are also less alloreactive as shown by a reduction in proliferation and cytokine production of responder cells in a mixed-lymphocyte reaction. This data further supports targeting CD6 as an effective means to inhibit pathogenic T cell activity in the treatment of autoimmune and inflammatory diseases.

Structure and function of DNA transposition assemblies involved in antibiotic resistance spreading

Transposons are mobile genetic elements that drive evolution and adaptation throughout the tree of life. In bacteria, they often transfer antibiotic resistance genes and contribute to the emergence of multidrug‐resistant pathogens.In this talk, I will present our recent discoveries on a group of transposons that efficiently propagate antibiotic resistance genes across diverse microbial communities. Using a dedicated computational pipeline, we mapped the most wide‐spread transposons in bacterial genomes and characterized their diversity, genetic cargo, and transfer dynamics. Selected elements were experimentally reconstituted to describe their molecular mechanisms and biochemical pathways. We determined numerous high‐resolution crystal and cryo‐EM structures of the protein‐DNA assemblies involved in transposon movement. The structures capture various stages of transposition and reveal an intricate interplay between the core transposition machinery and host‐encoded proteins. Protein interactions shape transposon and genomic DNA in unique ways to cut, exchange and re‐join strands in a tightly regulated manner. Importantly, our data also explain how specific DNA distortion and cleavage mechanisms enable the transposons to move between diverse genomic sites, expanding gene transfer across many species.These results shed new light onto the molecular strategies of antibiotic resistance carrying transposons and show how they evolved to effectively spread key genetic traits. Our insights also open new opportunities to develop strategies for blocking transposition and thereby help control resistance spreading.

Tuning riboflavin derivatives for photodynamic inactivation of pathogens

The development of effective pathogen reduction strategies is required due to the rise in antibiotic-resistant bacteria and zoonotic viral pandemics. Photodynamic inactivation (PDI) of bacteria and viruses is a potent reduction strategy that bypasses typical resistance mechanisms. Naturally occurring riboflavin has been widely used in PDI applications due to efficient light-induced reactive oxygen species (ROS) release. By rational design of its core structure to alter (photo)physical properties, we obtained derivatives capable of outperforming riboflavin’s visible light-induced PDI against E. coli and a SARS-CoV-2 surrogate, revealing functional group dependency for each pathogen. Bacterial PDI was influenced mainly by guanidino substitution, whereas viral PDI increased through bromination of the flavin. These observations were related to enhanced uptake and ROS-specific nucleic acid cleavage mechanisms. Trends in the derivatives’ toxicity towards human fibroblast cells were also investigated to assess viable therapeutic derivatives and help guide further design of PDI agents to combat pathogenic organisms.

Origin of iodine preferential attack at sulfur in phosphorothioate and subsequent P-O or P-S bond dissociation

Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (∼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.

Skeletal Ni electrode-catalyzed C-O cleavage of diaryl ethers entails direct elimination via benzyne intermediates

Diaryl ethers undergo electrocatalytic hydrogenolysis (ECH) over skeletal Ni cathodes in a mild, aqueous process that achieves direct C-O cleavage without initial benzene ring saturation. Mechanistic studies find that aryl phenyl ethers with a single para or meta functional group (methyl, methoxy, or hydroxy) are selectively cleaved to the substituted benzene and phenol, in contrast to recently reported homogeneous catalytic cleavage processes. Ortho positioning of substituents reverses this C-O bond selectivity, except for the 2-phenoxyphenol case. Together with isotope labeling and co-solvent studies, these results point to two distinct cleavage mechanisms: (a) dual-ring coordination and C-H activation, leading to vicinal elimination to form phenol and a surface-bound aryne intermediate which is then hydrogenated and released as the arene; and (b) surface binding in keto form by the phenolic ring of the hydroxy-substituted substrates, followed by direct displacement of the departing phenol. Notably, acetone inhibits the well-known reduction of phenol to cyclohexanol, affording control of product ring saturation. A byproduct of this work is the discovery that the ECH treatment completely defluorinates substrates bearing aromatic C-F and C-CF3 groupings.

Secondary structure RNA elements control the cleavage activity of DICER

The accurate and efficient cleavage of shRNAs and pre-miRNAs by DICER is crucial for their gene-silencing activity. Here, we conduct high-throughput DICER cleavage assays for more than ~20,000 different shRNAs and show the comprehensive cleavage activities of DICER on these sequences. We discover a single-nucleotide bulge (22-bulge), which facilitates the cleavage activity of DICER on shRNAs and human pre-miRNAs. As a result, this 22-bulge enhances the gene-silencing activity of shRNAs and the accuracy of miRNA biogenesis. In addition, various single-nucleotide polymorphism-edited 22-bulges are found to govern the cleavage sites of DICER on pre-miRNAs and thereby control their functions. Finally, we identify the single cleavage of DICER and reveal its molecular mechanism. Our findings improve the understanding of the DICER cleavage mechanism, provide a foundation for the design of accurate and efficient shRNAs for gene-silencing, and indicate the function of bulges in regulating miRNA biogenesis.

Cryo-EM structure of the prothrombin-prothrombinase complex

AbstractThe intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa–fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking.

The epidemic caused by the SARS-CoV-2 coronavirus, which has spread rapidly throughout the world, requires urgent and effective treatments considering that the appearance of viral variants limits the efficacy of vaccines. The main protease of SARS-CoV-2 (Mpro) is a highly conserved cysteine proteinase, fundamental for the replication of the coronavirus and with a specific cleavage mechanism that positions it as an attractive therapeutic target for the proposal of irreversible inhibitors. A structure-based strategy combining 3D pharmacophoric modeling, virtual screening, and covalent docking was employed to identify the interactions required for molecular recognition, as well as the spatial orientation of the electrophilic warhead, of various drugs, to achieve a covalent interaction with Cys145 of Mpro. The virtual screening on the structure-based pharmacophoric map of the SARS-CoV-2 Mpro in complex with an inhibitor N3 (reference compound) provided high efficiency by identifying 53 drugs (FDA and DrugBank databases) with probabilities of covalent binding, including N3 (Michael acceptor) and others with a variety of electrophilic warheads. Adding the energy contributions of affinity for non-covalent and covalent docking, 16 promising drugs were obtained. Our findings suggest that the FDA-approved drugs Vaborbactam, Cimetidine, Ixazomib, Scopolamine, and Bicalutamide, as well as the other investigational peptide-like drugs (DB04234, DB03456, DB07224, DB7252, and CMX-2043) are potential covalent inhibitors of SARS-CoV-2 Mpro.

Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2Apro and 3Cpro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2Apro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2Apro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2Apro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.

Mechanism and kinetics of 1‐phenyl‐1,2‐ethanediol cleavage catalyzed by Cu/Beta zeolite

AbstractTo explore the feasibility of producing high‐value products from biomass, the copper‐supported off‐Al H‐Beta zeolite was prepared and used as a catalyst. The 1‐phenyl‐1,2‐ethanediol was chosen as a catalyst, a model molecule of biomass, the reaction mechanism and reaction path of the oxidative cleavage were studied. The variation of concentration for different components and products distribution were obtained under different temperatures. Based on these experimental results, a kinetic model of the reaction system was established, and the kinetic parameters were developed through optimization algorithms. Results show that Brønsted acid and Lewis acid have a synergistic role in the oxidative cleavage of 1,2‐diol. The adding of Cu species can regulate the acidity and acid amount of the catalyst and can effectively improve the C─C bond breaking ability of the catalyst. 1‐phenyl‐1,2‐ethanediol firstly underwent oxidative cleavage to produce benzaldehyde, which further underwent parallel tandem reactions to get benzyl alcohol (BP), methyl benzoate (MB), and benzaldehyde dimethyl acetal (BDA), respectively. The highest activation energy is required for the oxidative cleavage reaction in the reaction system, and a higher temperature thus will be needed.

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

Porphyriynes: 18-π-Conjugated Macrocycles Incorporating a Triple Bond.

Tetraphenylporphyriyne (Pyne1), a novel porphyrin analogue with a C≡C bond incorporated into an 18-π-conjugated system, has been created via cleavage of the N-confused pyrrolic ring in Ag(III) N-confused tetraphenylporphyrin. The structure of Pyne1 was confirmed by X-ray crystallography and 1H NMR, IR, and UV-vis spectroscopy. The mechanism of cleavage of the N-confused pyrrolic ring was investigated by theoretical calculations. The successful synthesis of other Pynes indicated the generality of this protocol.

Effect of food processing on reduction and degradation pathway of pyrethroid pesticides in mackerel fillet (Scomberomorus commerson)

Pyrethroid contamination in fish can contribute to the dietary uptake of pesticides. To mitigate this risk, the effects of frozen storage, thermal treatments (boiling and grilling), and non-thermal treatments (pickling and curing) on the reduction of bifenthrin, cypermethrin, deltamethrin, and permethrin in mackerel fillets were investigated. The curing process was the most effective method that significantly depleted 74.82–79.45% of pyrethroid residues from fish fillets, followed by the synergistic effect of eight weeks’ frozen storage and grilling method (69.19–78.31%). Moreover, pyrethroid degradation pathways in processed fish were proposed into three major mechanisms of C1-C3 bond cleavage in cyclopropyl, dehalogenation, and double bond cleavage. These identical pathways incorporated with additional four mechanisms of dimerization, ester hydrolysis, oxidation, and reduction. This study recommended simple and effective processing practices for consumers and/or manufacturers to enhance food safety from the potential risks of consuming pyrethroid-contaminated fish.

Broken-Symmetry Density Functional Theory Analysis of the Ω Intermediate in Radical S-Adenosyl-l-methionine Enzymes: Evidence for a Near-Attack Conformer over an Organometallic Species.

Radical S-adenosyl-l-methionine (SAM) enzymes are found in all domains of life and catalyze a wide range of biochemical reactions. Recently, an organometallic intermediate, Ω, has been experimentally implicated in the 5'-deoxyadenosyl radical generation mechanism of the radical SAM superfamily. In this work, we employ broken-symmetry density functional theory to evaluate several structural models of Ω. The results show that the calculated hyperfine coupling constants (HFCCs) for the proposed organometallic structure of Ω are inconsistent with the experiment. In contrast, a near-attack conformer of SAM bound to the catalytic [4Fe-4S] cluster, in which the distance between the unique iron and SAM sulfur is ∼3 Å, yields HFCCs that are all within 1 MHz of the experimental values. These results clarify the structure of the ubiquitous Ω intermediate and suggest a paradigm shift reversal regarding the mechanism of SAM cleavage by members of the radical SAM superfamily.

Revealing G-lignin model compounds pyrolysis behavior: β-O-4 and 5-5′ dimer and trimer

It is anticipated that the insight into the formation of the pyrolysis products would contribute to a fundamental understanding of the bond cleavage mechanism. Herein, in order to have a better understanding of the lignin pyrolysis property, the lignin pyrolysis property was investigated by using multi-scale lignin model components. The TGA and Py-GC/MS data indicated that even the lignin trimer contains both of the β-O-4 and 5-5′ chemical structures can be cleaved simultaneously and multiple bond cleavage mechanisms come into play, any of which has an effect on each other. But, overall, the low pyrolysis temperature (450–550 °C) is conducive to the enrichment of guaiacols as the main bio-oil, especially for guaiacol. This evidence could provide theoretical support for the pyrolysis of softwood lignin and the guidance for industrial lignin's high-value utilization and directional conversion.

Histological and Immunohistochemical Studies to Determine the Mechanism of Cleft Palate Induction after Palatal Fusion in Mice Exposed to TCDD.

Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8–10-week-old ICR female mice. On day 12 of gestation, 40 μg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.

Pyridine coordination enabled stepwise PT/ET N-H transfer and metal-independent C-C cleavage mechanism for Cu-mediated dehydroacylation of unstrained ketones.

A density functional theory study of copper-mediated dehydroacylation of 4-phenyl-2-butanone to the corresponding olefin reveals a flexible N-H transfer process and a metal-independent C-C cleavage mechanism. When N'-methylpicolinohydrazonamide (MPHA) acts as the activating reagent, N-H cleavage can easily take place via stepwise proton transfer/electron transfer (PT/ET) and the rate-determining step is C-C homolysis with a total free energy barrier of 22.6 kcal mol-1, which is consistent with experimental observation of no kinetic isotope effects (KIE) at β-H. Besides, copper is found to have little influence on C-C cleavage, but is responsible for triggering single electron oxidation of the pre-aromatic intermediate (PAI). When replacing MPHA with picolinohydrazonamide (PHA), the second N-H transfer is 2.7 kcal mol-1 more favorable than C-C cleavage and dominates the pathway to aromatization, which explains there being no C-C cleavage product well. When N'-methylbenzohydrazonamide (MBHA) is adopted, the lack of pyridine coordination significantly reduces the stability of CuII and N-H transfer proceeds via a much more difficult proton coupled electron transfer (PCET) pathway, thus making N-H cleavage a rate-determining step with a total free energy barrier of up to 28.1 kcal mol-1.

Computational analysis of the mechanism of nonenzymatic peptide bond cleavage at the C-terminal side of an asparagine residue

Computational analysis of the mechanism of nonenzymatic peptide bond cleavage at the C-terminal side of an asparagine residue