Active Substrate Research Articles

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8 that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization invitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation invitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.

Structure-specific DNA endonuclease T7 endonuclease I cleaves DNA containing UV-induced DNA lesions.

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication and repair in Escherichia coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analysed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.

Plasma Treatment of Large-Area Polymer Substrates for the Enhanced Adhesion of UV-Digital Printing.

UV-digital printing belongs to the commonly used method for custom large-area substrate decoration. Despite low surface energy and adhesion, transparent polymer materials, such as polymethylmethacrylate (PMMA) and polycarbonate (PC), represent an ideal substrate for such purposes. The diffuse coplanar surface barrier discharge (DCSBD) in a novel compact configuration was used for substrate activation to improve ink adhesion to the polymer surface. This industrially applicable version of DCSBD was prepared, tested, and successfully implemented for the UV-digital printing process. Furthermore, wettability and surface free energy measurement, X-ray photoelectron spectroscopy, atomic force, and scanning electron microscopy evaluated the surface chemistry and morphology changes. The changes in the adhesion of the surface and of ink were analyzed by a peel-force and a crosscut test, respectively. A short plasma treatment (1-5 s) enhanced the substrate's properties of PMMA and PC while providing the pre-treatment suitable for further in-line UV-digital printing. Furthermore, we did not observe damage of or significant change in roughness affecting the substrate's initial transparency.

Low-Temperature Atmospheric Pressure Plasma Jets and their Applications in the Surface Activation of some Polymeric Substrates for Advanced Electronic Packaging

The present study applied low-temperature atmospheric plasma (LTAPP) jets to modify polymer substrates used in chip packaging. These substrates include bismaleimide triazine (BT), polyimide (PI) and PTFE materials. All these materials play important roles in modern semiconductor packaging. During the study, focuses were aimed the effects of reactive species (mainly OH and atomic oxygen) generated by atmospheric plasma jets on surface energy, water contact angle (WCA) and surface composition of these three substrates. In order to better understand and apply LTAPP jets, another focus will be on the understanding of the relationship among (1) the generation and distribution of various reactive species, (2) parameters for generating LTAPP jet, and (3) plasma characteristics. The plasma activation mechanism of these substrates was also discussed. In addition to plasma generation using AC high-voltage supplies, plasma characterization using optical emission spectroscopy (i.e., analysis of electron density, electron temperature, plasma temperature, and reactive species), XPS and water WCA analysis were used to understand surface activation mechanisms. In sum, Ar and Ar-O2 plasma jets can be used to modify the surfaces of these three substrates, although the latter relies on the generation of O radicals, while the former relies on OH radicals more. According to the results obtained by XPS analysis, the formation of C-O and C=O bond after plasma treatment is the main reason for surface activation in this study.

Mechanistic Insights into the electron‐transfer driven substrate activation by [4Fe‐4S]‐dependent Enzymes

Abstract[4Fe‐4S]‐dependent enzymes catalyze many different types of biological reactions. The quantum chemical cluster approach and the combined quantum mechanics/molecular mechanics approach, with the broken symmetry approach, are powerful tools for understanding the reaction mechanism in enzymes. This review discusses examples of the computational studies on [4Fe‐4S]‐dependent enzymes, focusing on the electron‐transfer driven substrate activation.

Interparticle Hydrogen Spillover in Enhanced Catalytic Reactions.

Interparticle hydrogen spillover is the phenomenon of H migration over different catalyst particles, which should be a physical mixture of at least two solid catalysts. In this review, we analyze examples of enhanced catalysis based on interparticle (reverse) hydrogen spillover. Simple physical mixtures of powdered catalysts containing metal catalysts of H2 dissociation/recombination and solid catalysts with active sites for substrate activation significantly enhance catalytic reactions. These reactions include aromatic hydrogenation, CO2 methanation, and the deoxydehydration of polyols, aromatization of lower paraffins, and direct coupling of benzene and alkanes. The acceleration effect and proposed reaction pathway of each example involving interparticle (reverse) hydrogen spillover are summarized. Simple reaction systems comprising physical mixtures of at least two powdery solid catalysts should enable unique catalysis in the future with the aid of interparticle (reverse) hydrogen spillover.

Enzymatic synthesis of tyrosol esters in organic solvents and ionic liquids: Correlation between enzyme activity and solvent properties

Tyrosol is a principle phenolic compound present in olive oil and wine with great pharmacological potentials due to its strong antioxidant property. Its application can be highly extended by increasing its lipophilicity through esterification. In this study, tyrosol esters were synthesized by enzymatic transesterification of tyrosol with vinyl esters, with a commercial lipase from Novozymes (Novozym 435, Candida antarctica lipase B (CALB) immobilized on macroporous acrylic resin beads) as the catalyst, both in organic solvents and in ionic liquids. Methyl tert-butyl ether (MTBE) was the best solvent, offering complete conversions towards the synthesis of tyrosol esters with varying chain lengths (C2-C18) within 1 h. Taking the enzymatic transesterification between tyrosol and vinyl acetate as the model reaction, 18 organic solvents and 24 ionic liquids were screened, and their solvent properties (i.e. hydrophobicity, polarity, viscosity, water activity, and partition coefficients of substrate and product) were correlated to the enzyme activity. Upon enzyme screening, four new lipases were found to be highly efficient, relative to the best-performed Novozym 435 and Lipozyme TLIM (Thermomyces lanuginosus lipase (TLL) immobilized on silica gel), in tyrosol ester synthesis. This work provides enzymatic methods of esterifying tyrosol that are much more efficient than those reported in literature, and will shed light on re-consideration of the roles of solvents in nonaqueous enzymology.

Zinc Oxide Tetrapods Doped with Silver Nanoparticles as a Promising Substrate for the Detection of Biomolecules via Surface-Enhanced Raman Spectroscopy

In this study, we report the fabrication and characterization of silver nanoparticle-doped zinc oxide tetrapod substrates used for surface-enhanced Raman scattering to detect rhodamine B. Prior to this, silver nanoparticle-doped zinc oxide tetrapods were synthesized using the solar physical vapor deposition method. Subsequently, silver-doped zinc oxide tetrapods were applied onto silicon wafers via the droplet evaporation process. The surface-enhanced Raman scattering activity of the silver nanoparticle-doped zinc oxide tetrapod substrate was evaluated by detecting rhodamine B using Raman spectroscopy. Our results demonstrate that the silver nanoparticle-doped zinc oxide tetrapod substrate exhibits surface-enhanced Raman scattering activity and can detect rhodamine B at concentrations as low as 3 μg/mL. This study suggests that silver nanoparticle-doped zinc oxide tetrapod substrates have potential as surface-enhanced Raman scattering platforms as well as potential for the detection of biomolecules.

Rationalizing the Regioselectivity of Azolation of Benzylic C–H Bonds under Photoredox Catalysis

AbstractA density functional theory (DFT) study was performed to evaluate the reaction mechanism of the C–N bond formation under an integrated hydrogen atom transfer/radical-polar crossover photoredox catalytic cycle. The regioselective activation of a model substrate, including three reactive positions (3° benzylic C–H bond, 2° benzylic C–H bond, and primary C–Cl bond) was addressed to distinguish among the radical C–H activation mechanism and the standard SN2 reaction. We demonstrated that activation of tertiary benzylic C–H bond is the most favored and forms exclusively the experimentally observed product. In addition, the whole photoredox catalytic cycle, including the outer-sphere electron-transfer steps, was characterized computationally.

Valorization of Lignocellulosic Molecules through Homogeneous Ru‐Catalyzed C−C and C−N Bond Forming Reactions

AbstractA sustainable upgrading of renewable bulk materials, obtained by decomposition of biomass lignocellulose, could facilitate their use in the industrial production of chemicals. Homogeneous ruthenium catalysis plays a fundamental role in this area. The review deals with the upgradation of lignocellulosic molecules through the formation of new C−C and C−N bonds. Selected examples of catalytic bond forming reactions reported until mid of 2023, include: (a) dehydrogenative based couplings of alcohols, (b) reductive amination of carbonyl compounds, (c) direct C−H functionalization of (hetero)aromatic compounds. These approaches fulfill the requirements of sustainability: application occurs without any previous substrate activation, thus reducing the production of waste. New chemicals containing the intrinsic molecular structure of lignocellulose substrates, showed a broad range of applications: green fuels, hydrogen carriers, monomers of polymers, intermediates of fine chemicals and pharmaceuticals. The results discussed in the review could inspire the reader to improve the valorization process, in terms of bio‐substrate scope as well as product complexity, through more strategic applications of the catalytic methods.

14N to 15N Isotopic Exchange of Nitrogen Heteroaromatics through Skeletal Editing.

The selective modification of nitrogen heteroaromatics enables the development of new chemical tools and accelerates drug discovery. While methods that focus on expanding or contracting the skeletal structures of heteroaromatics are emerging, methods for the direct exchange of single core atoms remain limited. Here, we present a method for 14N → 15N isotopic exchange for several aromatic nitrogen heterocycles. This nitrogen isotope transmutation occurs through activation of the heteroaromatic substrate by triflylation of a nitrogen atom, followed by a ring-opening/ring-closure sequence mediated by 15N-aspartate to effect the isotopic exchange of the nitrogen atom. Key to the success of this transformation is the formation of an isolable 15N-succinyl intermediate, which undergoes elimination to give the isotopically labeled heterocycle. These transformations occur under mild conditions in high chemical and isotopic yields.

Harmonizing Enzyme-like Cofactors to Boost Nanozyme Catalysis.

Engineering isolated metal sites resembling the primary coordination sphere of metallocofactors enables atomically dispersed materials as promising nanozymes. However, most existing nanozymes primarily focus on replicating specific metallocofactors while neglecting other supporting cofactors within active pockets, leading to reduced electron transfer (ET) efficiency and thus inferior catalytic performances. Herein, we report a metal-organic framework UiO-67 nanozyme with atomically dispersed iron sites, which involves multiple tailored enzyme-like nanocofactors that synergistically drive the ET process for enhanced peroxidase-like catalysis. Among them, the linker-coupled atomic iron site plays a critical role in substrate activation, while bare linkers and zirconia nodes facilitate the ET efficiency of intermediates. The synergy of three nanocofactors results in a 4.29-fold enhancement compared with the single effort of isolated metal site-based nanocofactor, holding promise in immunoassay for sensitive detection of chlorpyrifos. This finding opens a new way for designing high-performance nanozymes by harmonizing various nanocofactors at the atomic and molecular scale.

Harmonizing Enzyme‐like Cofactors to Boost Nanozyme Catalysis

AbstractEngineering isolated metal sites resembling the primary coordination sphere of metallocofactors enables atomically dispersed materials as promising nanozymes. However, most existing nanozymes primarily focus on replicating specific metallocofactors while neglecting other supporting cofactors within active pockets, leading to reduced electron transfer (ET) efficiency and thus inferior catalytic performances. Herein, we report a metal–organic framework UiO‐67 nanozyme with atomically dispersed iron sites, which involves multiple tailored enzyme‐like nanocofactors that synergistically drive the ET process for enhanced peroxidase‐like catalysis. Among them, the linker‐coupled atomic iron site plays a critical role in substrate activation, while bare linkers and zirconia nodes facilitate the ET efficiency of intermediates. The synergy of three nanocofactors results in a 4.29‐fold enhancement compared with the single effort of isolated metal site‐based nanocofactor, holding promise in immunoassay for sensitive detection of chlorpyrifos. This finding opens a new way for designing high‐performance nanozymes by harmonizing various nanocofactors at the atomic and molecular scale.

Single Cobalt Ion-Immobilized Covalent Organic Framework for Lithium-Sulfur Batteries with Enhanced Rate Capabilities.

Covalent organic frameworks (COFs) are notable for their remarkable structure, function designability, and tailorability, as well as stability, and the introduction of "open metal sites" ensures the efficient binding of small molecules and activation of substrates for heterogeneous catalysis and energy storage. Herein, we use the postsynthetic metal sites to catalyze polysulfide conversion and to boost the binding affinity to active matter for lithium-sulfur batteries (LSBs). A dual-pore COF, USTB-27, with hxl topology has been successfully assembled from the imine chemical reaction between 2,3,8,9,14,15-hexa(4-formylphenyl)diquinoxalino [2,3-a:2',3'-c]phenazine and [2,2'-bipyridine]-5,5'-diamine. The chelating nitrogen sites of both modules are able to postsynthetically functionalize with single cobalt sites to generate USTB-27-Co. The discharge capacity of the sulfur-loaded S@USTB-27-Co composite in a LSB is 1063, 945, 836, 765, 696, and 644 mA h g-1 at current densities of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 C, respectively, much superior to that of non-cobalt-functionalized species S@USTB-27. Following the increased current densities, the rate performance of S@USTB-27-Co is much better than that of S@USTB-27. In particular, the capacity retention at 5.0 C has a magnificent increase from 19% for the latter species to 61% for the former one. Moreover, S@USTB-27-Co exhibits a higher specific capacity of 543 mA h g-1 than that of S@USTB-27 (402 mA h g-1) at a current density of 1.0 C after electrochemical cycling for 500 runs. This work illustrates the "open metal sites" strategy to engineer the active chemical component conversion in COF channels as well as their binding strength for specific applications.

Selective Electrosynthesis of Urea via C−N Coupling: Current Status, Challenges and Future Prospects

AbstractSelective electrosynthesis of urea from carbon source (CO2) and nitrogen (N2, NO3−, NO2−) source provides a sustainable strategy for production of essential nitrogen‐based fertilizers. The traditional technology of urea synthesis employs two consecutive processes, synthesis of NH3 from N2+H2 and urea from NH3+CO2, which always involves high energy consumption and environmental concerns. By contrast, urea electrosynthesis avoids the above harsh steps and meanwhile has higher atomic economy. Herein, we present a comprehensive review on the progress and prospect of urea electrosynthesis from abundant carbon and nitrogen sources via coupling of C−N under ambient conditions, and also discuss the mechanism researches, strategies of substrates activation, current challenges and future opportunities of urea electrosynthesis. The urea electrosynthesis via C−N coupling realizes abundant and low‐cost inorganic carbon and nitrogen sources for efficient resource utilization and provides guidance and reference for C−C, C−S or other coupling reactions.

Oxidative Dearomatization of Pyridines.

Dearomatization of pyridines is a well-established synthetic approach to access piperidines. Although remarkably powerful, existing dearomatization processes have been limited to the hydrogenation or addition of carbon-based nucleophiles to activated pyridiniums. Here, we show that arenophile-mediated dearomatizations can be applied to pyridines to directly introduce heteroatom functionalities without prior substrate activation. The arenophile platform in combination with olefin oxidation chemistry provides access to dihydropyridine cis-diols and epoxides. These previously elusive compounds are now readily accessible and can be used for the downstream preparation of diversely functionalized piperidines.

Recombinant production and characterization of a metal ion-independent Lysophospholipase from a hyperthermophilic archaeon Pyrococcus abyssi DSM25543

Genome sequence of Pyrococcus abyssi DSM25543 contains a coding sequence (PAB_RS01410) for α/β hydrolase (WP_010867387.1). Structural analysis revealed the presence of a consensus motif GXSXG and a highly conserved catalytic triad in the amino acid sequence of α/β hydrolase that were characteristic features of lysophospholipases. A putative lysophospholipase from P. abyssi with its potential applications in oil degumming and starch processing was heterologously produced in E. coli Rosetta (DE3) pLysS in soluble form followed by its purification and characterization. The recombinant enzyme was found to be active at temperature of 40–90 °C and pH 5.5–7.0. However, the enzyme exhibited its optimum activity at 65 °C and pH 6.5. None of the metal ions (Mn2+, Mg2+, Ni2+, Cu2+, Fe2+, Co2+, Zn2+ and Ca2+) being tested had stimulatory effect on lysophospholipase activity. Km and Vmax for hydrolysis of 4-nitrophenyl butyrate were calculated to be 1 ± 0.089 mM and 1637 ± 24.434 U/mg, respectively. It is the first report on the soluble production and characterization of recombinant lysophospholipase from P. abyssi which exhibits its lipolytic activity in the absence of divalent metal ions. Broad substrate specificity, activity and stability at elevated temperatures make recombinant lysophospholipase an ideal candidate for potential industrial applications.

Silver nanoparticles modified sulfur-containing POSS polymer membrane substrate for adsorption and surface-enhanced Raman scattering analysis of chrysoidine in food samples

In analysis of complex samples, the stability and sensitivity of surface-enhanced Raman scattering (SERS) substrates may be compromised by matrix interference. To address this issue, a membrane substrate was prepared for fast enrichment, separation, and detection of chrysoidine all-in-one. The silver nanoparticles modified sulfur-containing POSS polymer (AgNPs/POSS-P-S) SERS membrane substrate was fabricated using polyhedral oligomeric silsesquioxane (POSS) as support materials. Through in-situ growth, AgNPs were uniformly modified on POSS-P-S to ensure the stability and SERS activity of the membrane substrate. The enhancement factor of the malachite green was up to 5.3 × 105. By loading the AgNPs/POSS-P-S on membrane, on the other hand, the SERS membrane substrate can also serve as an adsorption medium for separating chrysoidine from sample matrix. Furthermore, the specific sensing mechanism of AgNPs/POSS-P-S for chrysoidine was investigated and a fast, sensitive, and selective method for its quantification was established, with a linear range of 0.010–2.0 mg/L and the limits of detection at 3.7 μg/L. In addition, the SERS method was successfully applied for the analysis of chrysoidine in beverages and chili products with the recoveries in the range of 83.5%–113.4 % and the relative standard deviations in 3.2%–9.0 %. The proposed AgNPs/POSS-P-S membrane based SRES method has great potential for rapid chrysoidine analysis in food samples.

Identification of Factor XIII β-Sandwich Residues Mediating Glutamine Substrate Binding and Activation Peptide Cleavage.

Factor XIII (FXIII) forms covalent crosslinks across plasma and cellular substrates and has roles in hemostasis, wound healing, and bone metabolism. FXIII activity is implicated in venous thromboembolism (VTE) and is a target for developing pharmaceuticals, which requires understanding FXIII - substrate interactions. Previous studies proposed the β-sandwich domain of the FXIII A subunit (FXIII-A) exhibits substrate recognition sites. Recombinant FXIII-A proteins (WT, K156E, F157L, R158Q/E, R171Q, and R174E) were generated to identify FXIII-A residues mediating substrate recognition. Proteolytic (FXIII-A*) and non-proteolytic (FXIII-A°) forms were analyzed for activation and crosslinking activities toward physiological substrates using SDS-PAGE and MALDI-TOF MS. All FXIII-A* variants displayed reduced crosslinking abilities compared to WT for Fbg αC (233 - 425), fibrin, and actin. FXIII-A* WT activity was greater than A°, suggesting the binding site is more exposed in FXIII-A*. With Fbg αC (233 - 425), FXIII-A* variants R158Q/E, R171Q, and R174E exhibited decreased activities approaching those of FXIII-A°. However, with a peptide substrate, FXIII-A* WT and variants showed similar crosslinking suggesting the recognition site is distant from the catalytic site. Surprisingly, FXIII-A R158E and R171Q displayed slower thrombin activation than WT, potentially due to loss of crucial H-bonding with neighboring activation peptide (AP) residues. In conclusion, FXIII-A residues K156, F157, R158, R171, and R174 are part of a binding site for physiological substrates [fibrin (α and γ) and actin]. Moreover, R158 and R171 control AP cleavage during thrombin activation. These investigations provide new molecular details on FXIII - substrate interactions that control crosslinking abilities.

Study of the Antibacterial Activity of Superhydrophilic and Superhydrophobic Copper Substrates against Multi-Drug-Resistant Hospital-Acquired Pseudomonas aeruginosa Isolates.

The global spread of multidrug-resistant (MDR) hospital-acquired pathogens is a serious problem for healthcare units. The challenge of the spreading of nosocomial infections, also known as hospital-acquired pathogens, including Pseudomonas aeruginosa, must be addressed not only by developing effective drugs, but also by improving preventive measures in hospitals, such as passive bactericidal coatings deposited onto the touch surfaces. In this paper, we studied the antibacterial activity of superhydrophilic and superhydrophobic copper surfaces against the P. aeruginosa strain PA103 and its four different polyresistant clinical isolates with MDR. To fabricate superhydrophilic and superhydrophobic coatings, we subjected the copper surfaces to laser processing with further chemosorption of fluorooxysilane to get a superhydrophobic substrate. The antibacterial activity of superhydrophilic and superhydrophobic copper surfaces was shown, with respect to both the collection strain PA103 and polyresistant clinical isolates of P. aeruginosa, and the evolution of the decontamination of a bacterial suspension is presented and discussed. The presented results indicate the promising potential of the exploitation of superhydrophilic coatings in the manufacture of contact surfaces for healthcare units, where the risk of infection spread and contamination by hospital-acquired pathogens is extremely high.