Covalent Binding Sites Research Articles

Target Fishing Reveals a Novel Mechanism of N-Acylamino Saccharin Derivatives Targeting Glyceraldehyde-3-Phosphate Dehydrogenase toward Cyanobacterial Blooms Control.

Based on current challenges of poor targeting and limited choices in chemical control methods of cyanobacterial blooms (CBs), identifying new targets is an urgent and formidable task in the quest for target-based algaecides. This study discovered N-acylamino saccharin derivatives exhibiting potent algicidal activity. Thus, using N-acylamino saccharin as the probes, glyceraldehyde-3-phosphate dehydrogenase from cyanobacterial (CyGAPDH) was identified as a new target of algaecides through the activity-based protein profiling (ABPP) strategy for the first time. Building upon the structure of Probe2, a series of derivatives were designed and synthesized, with compound b6 demonstrating the most potent inhibitory activity against CyGAPDH and Synechocystis sp. PCC6803 (IC50 = 1.67 μM and EC50 = 1.15 μM). Furthermore, the potential covalent binding model of b6 to the cysteine residue C154 was explored through covalent possibility prediction, LC-MS experiments, substrate competitive inhibition experiments, and molecular docking. Especially, the results revealed C154 as a crucial covalent binding site, with residues T184 and R11 forming robust hydrophobic interactions and H181 establishing significant hydrogen-bonding interactions with b6, highlighting their potential as essential pharmacophores. In summary, this study not only identifies a novel target of algaecides for the control of CB but also lays the solid foundation for the development of targeted covalent algaecides.

Discovery and characterization of naturally occurring covalent inhibitors of SARS-CoV-2 Mpro from the antiviral herb Ephedra

The Chinese herb Ephedra (also known as Mahuang) has been extensively utilized for the prevention and treatment of coronavirus-induced diseases, including coronavirus disease 2019 (COVID-19). However, the specific anti-SARS-CoV-2 compounds and mechanisms have not been fully elucidated. The main protease (Mpro) of SARS-CoV-2 is a highly conserved enzyme responsible for proteolytic processing during the viral life cycle, making it a critical target for the development of antiviral therapies. This study aimed to identify naturally occurring covalent inhibitors of SARS-CoV-2 Mpro from Ephedra and to investigate their covalent binding sites. The results demonstrated that the non-alkaloid fraction of Ephedra (ENA) exhibited a potent inhibitory effect against the SARS-CoV-2 Mpro effect, whereas the alkaloid fraction did not. Subsequently, the chemical constituents in ENA were identified, and the major constituents’ anti-SARS-CoV-2 Mpro effects were evaluated. Among the tested constituents, herbacetin (HE) and gallic acid (GA) were found to inhibit SARS-CoV-2 Mpro in a time- and dose-dependent manner. Their combination displayed a significant synergistic effect on this key enzyme. Additionally, various techniques, including inhibition kinetic assays, chemoproteomic methods, and molecular dynamics simulations, were employed to further elucidate the synergistic anti-Mpro mechanisms of the combination of HE and GA. Overall, this study deciphers the naturally occurring covalent inhibitors of SARS-CoV-2 Mpro from Ephedra and characterizes their synergistic anti-Mpro synergistic effect, providing robust evidence to support the anti-coronavirus efficacy of Ephedra.

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Glucopeptide Superstructure Hydrogel Promotes Surgical Wound Healing Following Neoadjuvant Radiotherapy by Producing NO and Anticellular Senescence.

Neoadjuvant radiotherapy, a preoperative intervention regimen for reducing the stage of primary tumors and surgical margins, has gained increasing attention in the past decade. However, radiation-induced skin damage during neoadjuvant radiotherapy exacerbates surgical injury, remarkably increasing the risk of refractory wounds and compromising the therapeutic effects. Radiation impedes wound healing by increasing the production of reactive oxygen species and inducing cell apoptosis and senescence. Here, a self-assembling peptide (R-peptide) and hyaluronic-acid (HA)-based and cordycepin-loaded superstructure hydrogel is prepared for surgical incision healing after neoadjuvant radiotherapy. Results show that i) R-peptide coassembles with HA to form biomimetic fiber bundle microstructure, in which R-peptide drives the assembly of single fiber through π-π stacking and other forces and HA, as a single fiber adhesive, facilitates bunching through electrostatic interactions. ii) The biomimetic superstructure contributes to the adhesion and proliferation of cells in the surgical wound. iii) Aldehyde-modified HA provides dynamic covalent binding sites for cordycepin to achieve responsive release, inhibiting radiation-induced cellular senescence. iv) Arginine in the peptides provides antioxidant capacity and a substrate for the endogenous production of nitric oxide to promote wound healing and angiogenesis of surgical wounds after neoadjuvant radiotherapy.

Time-series analysis of rhenium(I) organometallic covalent binding to a model protein for drug development.

Metal-based complexes with their unique chemical properties, including multiple oxidation states, radio-nuclear capabilities and various coordination geometries yield value as potential pharmaceuticals. Understanding the interactions between metals and biological systems will prove key for site-specific coordination of new metal-based lead compounds. This study merges the concepts of target coordination with fragment-based drug methodologies, supported by varying the anomalous scattering of rhenium along with infrared spectroscopy, and has identified rhenium metal sites bound covalently with two amino acid types within the model protein. A time-based series of lysozyme-rhenium-imidazole (HEWL-Re-Imi) crystals was analysed systematically over a span of 38 weeks. The main rhenium covalent coordination is observed at His15, Asp101 and Asp119. Weak (i.e. noncovalent) interactions are observed at other aspartic, asparagine, proline, tyrosine and tryptophan side chains. Detailed bond distance comparisons, including precision estimates, are reported, utilizing the diffraction precision index supplemented with small-molecule data from the Cambridge Structural Database. Key findings include changes in the protein structure induced at the rhenium metal binding site, not observed in similar metal-free structures. The binding sites are typically found along the solvent-channel-accessible protein surface. The three primary covalent metal binding sites are consistent throughout the time series, whereas binding to neighbouring amino acid residues changes through the time series. Co-crystallization was used, consistently yielding crystals four days after setup. After crystal formation, soaking of the compound into the crystal over 38 weeks is continued and explains these structural adjustments. It is the covalent bond stability at the three sites, their proximity to the solvent channel and the movement of residues to accommodate the metal that are important, and may prove useful for future radiopharmaceutical development including target modification.

New Means and Challenges in the Targeting of BTK.

Bruton's tyrosine kinase (BTK) is central to the survival of malignant and normal B lymphocytes and has been a crucial therapeutic target of several generations of kinase inhibitors and newly developed degraders. These new means for targeting BTK have added additional agents to the armamentarium for battling cancers dependent on B-cell receptor (BCR) signaling, including chronic lymphocytic leukemia and other non-Hodgkin lymphomas. However, the development of acquired resistance mutations to each of these classes of BTK inhibitors has led to new challenges in targeting BTK as well as novel insights into BCR signaling. The first-generation covalent BTK inhibitor ibrutinib is susceptible to mutations affecting the covalent binding site, cysteine 481 (C481). Newer noncovalent BTK inhibitors, such as pirtobrutinib, overcome C481 mutation-mediated resistance but are susceptible to other kinase domain mutations, particularly at residues Threonine 474 and Leucine 528. In addition, these novel BTK inhibitor resistance mutations have been shown biochemically and in patients to cause cross-resistance to some covalent BTK inhibitors. Importantly, newer generation covalent BTK inhibitors zanubrutinib and acalabrutinib are susceptible to the same mutations that confer resistance to noncovalent inhibitors. The BTK L528W mutation is of particular interest as it disrupts the kinase activity of BTK, rendering it kinase dead. This observation suggests that BTK may act independently of its kinase activity as a scaffold. Thus, the timely development of BTK degrading proteolysis targeting drugs has allowed for degradation, rather than just enzymatic inhibition, of BTK in B-cell lymphomas, and early clinical trials to evaluate BTK degraders are underway.

A Freestanding, Dissolution- and Diffusion-Limiting, Flexible Sulfur Electrode Enables High Specific Capacity at High Mass Loading.

The acquisition of stable and high-areal-capacity S cathodes over 10mA h cm-2 is a critical and indispensable step to realize the high energy density configuration. However, increasing the areal capacity of S cathodes often deteriorates the specific capacity and stability due to the aggravated dissolution of S and diffusion of solvable polysulfides in the thick electrode. Herein, the design of a freestanding composite cathode that leverages 3D covalent binding sites and chemical adsorption environment to offer dissolution-limiting and diffusion-blocking functions of S species is reported. By employing this architecture, the coin cell exhibits excellent cycling stability and an exceptional specific capacity of 1444.3mA h g-1 (13mA h cm-2), and the pouch cell configuration manifests a noteworthy areal capacity exceeding 11mA h cm-2. This performance is coupled with excellent flexibility, demonstrated through consecutive bending cycle tests, even at a sulfur loading of 9.00mg cm-2. This study lays the foundation for the development of flexible Li-S batteries with increased loading capacities and exceptional performance.

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors.

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

Detection of H. pylori outer membrane protein (HopQ) biomarker using electrochemical impedimetric immunosensor with polypyrrole nanotubes and carbon nanotubes nanocomposite on screen-printed carbon electrode

Helicobacter pylori (H. pylori) is classified as a class I carcinogen that colonizes the human gastrointestinal (GI) tract. The detection at low concentrations is crucial in combatting H. pylori. HopQ protein is located on H. pylori's outer membrane and is expressed at an early stage of contamination, which signifies it as an ideal biomarker. In this study, we presented the development of an electrochemical impedimetric immunosensor for the ultra-sensitive detection of HopQ at low concentrations. The sensor employed polypyrrole nanotubes (PPy-NTs) and carboxylated multi-walled carbon nanotubes (MWCNT-COOH) nanocomposite. PPy-NTs were chosen for their excellent conductivity, biocompatibility, and redox capabilities, simplifying sample preparation by eliminating the need to add redox probes upon measurement. MWCNT-COOH provided covalent binding sites for HopQ antibodies (HopQ-Ab) on the biosensor surface. Characterization of the biosensor was performed using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), contact angle measurements, and electrochemical impedance spectroscopy (EIS), complemented by numerical semiempirical quantum calculations. The results demonstrated a dynamic linear range of 5 pg/mL to 1.063 ng/mL and an excellent selectivity, with the possibility of excluding interference using EIS data, specifically charge transfer resistance and double-layer capacitance as multivariants for the calibration curve. Using two EIS components, the limit of detection is calculated to be 2.06 pg/mL. The biosensor was tested with a spiked drinking water sample and showed a signal recovery of 105.5% when detecting 300 pg/mL of HopQ. This novel H. pylori biosensor offers reliable, simple, portable, and rapid screening of the bacteria.

Tartary buckwheat protein-phenol conjugate prepared by alkaline-based environment: Identification of covalent binding sites of phenols and alterations in protein structural and functional characteristics

Tartary buckwheat protein-rutin/quercetin covalent complex was synthesized in alkaline oxygen-containing environment, and its binding sites, conformational changes and functional properties were evaluated by multispectral technique and proteomics. The determination of total sulfhydryl and free amino groups showed that rutin/quercetin can form a covalent complex with BPI and could significantly reduce the group content. Ultraviolet-visible spectrum analysis showed that protein could form new characteristic peaks after binding with rutin/quercetin. Circular dichroism spectrum analysis showed that rutin and quercetin caused similar changes in the secondary structure of proteins, both promoting β-sheet to α-helix, β-ture and random coil transformation. The fluorescence spectrometry results showed that the combination of phenols can cause the fluorescence quenching, and the combination of rutin was stronger than the quercetin. Proteomics showed that there were multiple covalent binding sites between phenols and protein. Rutin had a high affinity for arginine, and quercetin and cysteine had high affinity. Meanwhile, the combination of rutin/quercetin and protein had reduced the surface hydrophobic ability of the protein, and improved the foaming, stability and antioxidant properties of the protein. This study expounded the mechanism of the combination of BPI and rutin/quercetin, and analysed the differences of the combination of protein and phenols in different structures. The findings can provide a theoretical basis for the development of complexes in the area of food.

Neuromorphic sensing of biomolecules covalently immobilised on polydimethyl glutarimide

Polydimethyl glutarimide (PMGI) layers with sub-micron thicknesses have been modified in a 2.5 kV Ar plasma immersion ion implantation (PIII) process to introduce free radical covalent binding sites. The surface roughness of the PMGI increased after the PIII treatment but no through-layer defects were observed. When applied to the treated PMGI, horseradish peroxidase (HRP) enzyme remained bound to the surface after extended immersion in sodium dodecyl sulfate solution (SDS). Hence, covalent binding between the activated surface and enzyme was confirmed. This covalent binding was achieved up to 24-h after the PIII process. The treated PMGI was then incorporated as a gate dielectric layer within a lateral three-terminal electrolyte-gated device. The device output characteristics resembled those of post-synaptic outputs; as successive (pre-synaptic) voltage pulses were applied to the gate, paired pulse depression and spike rate dependent plasticity were observed in the source-drain (post-synaptic) current. These characteristics were altered by the presence of HRP immobilised on the plasma-modified PMGI gate dielectric layer thus providing readout detection. These results and preliminary device characteristics show the potential for the plasma functionalized PMGI as a sensitive and reproducible biosensing technology.

Dynamically crosslinked thermoresponsive granular hydrogels.

Granular hydrogels are a promising biomaterial for a wide range of biomedical applications, including tissue regeneration, drug/cell delivery, and 3D printing. These granular hydrogels are created by assembling microgels through the jamming process. However, current methods for interconnecting the microgels often limit their use due to the reliance on postprocessing for crosslinking through photoinitiated reactions or enzymatic catalysis. To address this limitation, we incorporated a thiol-functionalized thermo-responsive polymer into oxidized hyaluronic acid microgel assemblies. The rapid exchange rate of thiol-aldehyde dynamic covalent bonds allows the microgel assembly to be shear-thinning and self-healing, with the phase transition behavior of the thermo-responsive polymer serving as secondary crosslinking to stabilize the granular hydrogels network at body temperature. This two-stage crosslinking system provides excellent injectability and shape stability, while maintaining mechanical integrity. In addition, the aldehyde groups of the microgels act as covalent binding sites for sustained drug release. These granular hydrogels can be used as scaffolds for cell delivery and encapsulation, and can be 3D printed without the need for post-printing processing to maintain mechanical stability. Overall, our work introduces thermo-responsive granular hydrogels with promising potential for various biomedical applications.

Investigating structural features of dimeric SARS-CoV-2 Mpro catalytic site with bound covalent ligands at physiological temperature

The SARS-CoV-2 main protease (Mpro) plays an important role in the viral transcription and replication of the SARS-CoV-2 virus that is causing the Covid-19 pandemic worldwide. Therefore, it represents a very attractive target for drug development for treatment of this disease. It is a cysteine protease because it has in the active site the catalytic dyad composed of cysteine (C145) and histidine (H41). The catalytic site represents the binding site for inhibitors, many of them bind to Mpro with a covalent bond. In this research, structural and physiochemical characteristics of the Mpro binding site are investigated when the ligand 11a is covalently and non-covalently bound. All-atom molecular dynamics (MD) simulations were run for 500 ns at physiological temperature (310 K). It is found that conformations of both the Mpro protein and the ligand are stable during the simulation with covalently bound complex showing stronger stability. When the ligand is covalently bound (its final state), residues that stably interact with the ligand are H41, C145, H163, H164 and E166. The optimal conformation of these residues is stabilized also via the Hbond interactions with the catalytic water present in the Mpro binding site. In the case of the non-covalently bound ligand (state before the covalent bond is formed), the binding site residues retain their conformations similar to the covalent binding site, and they still form Hbonds with the catalytic water, except H41. This residue, instead, adopts a different conformation and looses the Hbond with the catalytic water, leaving more freedom to move to the ligand. We hypothesize that H41 could play a role in guiding the ligand to the optimal position for final covalent bonding. Further analyses are in process to check this hypothesis. These results represent an important basis for studying drug candidates against SARS-CoV-2 by means of computer aided drug design.

In Vivo MRI Tracking of Degradable Polyurethane Hydrogel Degradation In Situ Using a Manganese Porphyrin Contrast Agent.

A noninvasive method to track implanted biomaterials is desirable for real-time monitoring of material interactions with host tissues and assessment of efficacy and safety. To explore quantitative in vivo tracking of polyurethane implants using a manganese porphyrin (MnP) contrast agent containing a covalent binding site for pairing to polymers. Prospective, longitudinal. Rodent model of dorsal subcutaneous implants (10 female Sprague Dawley rats). A 3-T; two-dimensional (2D) T1-weighted spin-echo (SE), T2-weighted turbo SE, three-dimensional (3D) spoiled gradient-echo T1 mapping with variable flip angles. A new MnP-vinyl contrast agent to covalently label polyurethane hydrogels was synthesized and chemically characterized. Stability of binding was assessed in vitro. MRI was performed in vitro on unlabeled hydrogels and hydrogels labeled at different concentrations, and in vivo on rats with unlabeled and labeled hydrogels implanted dorsally. In vivo MRI was performed at 1, 3, 5, and 7 weeks postimplantation. Implants were easily identified on T1-weighted SE, and fluid accumulation from inflammation was distinguished on T2-weighted turbo SE. Implants were segmented on contiguous T1-weighted SPGR slices using a threshold of 1.8 times the background muscle signal intensity; implant volume and mean T1 values were then calculated at each timepoint. Histopathology was performed on implants in the same plane as MRI and compared to imaging results. Unpaired t-tests and one-way analysis of variance (ANOVA) were used for comparisons. A P value <0.05 was considered to be statistically significant. Hydrogel labeling with MnP resulted in a significant T1 reduction in vitro (T1=517 ± 36 msec vs. 879 ± 147 msec unlabeled). Mean T1 values of labeled implants in rats increased significantly by 23% over time, from 1 to 7 weeks postimplantation (651 ± 49 msec to 801 ± 72 msec), indicating decreasing implant density. Polymer-binding MnP enables in vivo tracking of vinyl-group coupling polymers. 1. Stage 1.

Discovery of Isojacareubin as a covalent inhibitor of SARS-CoV-2 main protease using structural and experimental approaches.

The ongoing pandemic with the emergence of immune evasion potential and, particularly, the current omicron subvariants intensified the situation further. Although vaccines are available, the immune evasion capabilities of the recent variants demand further efficient therapeutic choices to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Hence, considering the necessity of the small molecule inhibitor, we target the main protease (3CLpro), which is an appealing target for the development of antiviral drugs against SARS-CoV-2. High-throughput molecular in silico screening of South African natural compounds database reported Isojacareubin and Glabranin as the potential inhibitors for the main protease. The calculated docking scores were reported to be -8.47 and -8.03 kcal/mol, respectively. Moreover, the structural dynamic assessment reported that Isojacareubin in complex with 3CLpro exhibit a more stable dynamic behavior than Glabranin. Inhibition assay indicated that Isojacareubin could inhibit SARS-CoV-2 3CLpro in a time- and dose-dependent manner, with half maximal inhibitory concentration values of 16.00 ± 1.35 μM (60 min incubation). Next, the covalent binding sites of Isojacareubin on SARS-CoV-2 3CLpro was identified by biomass spectrometry, which reported that Isojacareubin can covalently bind to thiols or Cysteine through Michael addition. To evaluate the inactivation potency of Isojacareubin, the inactivation kinetics was further investigated. The inactivation kinetic curves were plotted according to various concentrations with gradient-ascending incubation times. The KI value of Isojacareubin was determined as 30.71 μM, whereas the Kinact value was calculated as 0.054 min-1 . These results suggest that Isojacareubin is a covalent inhibitor of SARS-CoV-2 3CLpro .

Mussel-Inspired Electro-oxidation-Modified Three-Dimensional Printed Carriers for a Versatile Enzyme Immobilization Approach

Conventional enzyme immobilization approaches can only immobilize certain specific enzymes with poor generality. Attempts to improve the universality of enzyme types tend to impart them with more enzymatic catalysis applications. Here, inspired by mussel adhesive proteins, we present a novel eco-friendly surface carrier that was 3D printed and modified by electro-oxidation for enzyme immobilization. The carrier was fabricated through 3D printing by transforming acrylonitrile butadiene styrene (ABS) material into a suitable structure (3DABS). Then, electro-oxidative modification was performed on the surface to form a polydopamine (PDA) coating (3DABS-PDA). The desired structures for the enzyme immobilization carriers were obtained through 3D printing technology, while electro-oxidation modification of the surface provided numerous and firmly covalent binding sites. Based on these features, we have demonstrated that 3D printed and electro-oxidation-modified carriers could be applied to immobilize different types of enzymes. The loading capacity of all immobilized enzymes (galV, EG5C-1, XynLK9, and kdcA) exceeded 25 mg·g–1 (37.7 mg·g–1 for galV), and after 10 reuse cycles, the substrate conversion rate of 3DABS-PDA@galV was still over 85%. The carriers can be reused after simple processing. These results indicate that 3DABS-PDA provides an efficient, sustainable, and versatile approach for enzyme immobilization and exhibits excellent value in various enzymatic catalysis applications.

Soy protein–phlorizin conjugate prepared by tyrosinase catalysis: Identification of covalent binding sites and alterations in protein structure and functionality

Tyrosinase-catalyzed synthesis of soy 7S/11S–phlorizin conjugates was performed, and the reaction sites, conformation alterations and functional properties of complexes were evaluated using proteomic, in combination with multispectral technologies. Phlorizin was conjugated to 7S/11S primarily via residues of Lys, Cys, His and Arg residues. The phlorizin binding equivalents and decreased contents of free and total sulfhydryl groups and free amino groups confirmed the covalent interaction in the 7S/11S–phlorizin complexes. Conjugation with phlorizin promoted the conversion of α-helix to β-sheet and β-turn, with simultaneous transformation of the microenvironments around Trp and Tyr residues to hydrophilic and hydrophobic microenvironments, respectively, and lowering of the surface hydrophobicity of 7S/11S. The DPPH and ABTS radical scavenging abilities and α-glucosidase inhibitory activities of 7S/11S were increased by three–, two– and three–fold after the covalent binding of phlorizin. The study provided an ideal tyrosinase-catalyzed approach to fabricate custom–tailored nutritional soy protein–polyphenol products.

Efficacy of the Immobilized Kocuria flava Lipase on Fe3O4/Cellulose Nanocomposite for Biodiesel Production from Cooking Oil Wastes

The increasing global demand for petroleum oils has led to a significant increase in their cost and has led to the search for renewable alternative waste resources for biodiesel synthesis and production using novel environmentally sound and acceptable methods. In the current study, Kocuria flava lipase was immobilized on Fe3O4/cellulose nanocomposite; and used as a biocatalyst for the conversion of cooking oil wastes into biodiesel through the transesterification/esterification process. The characterization of Fe3O4/cellulose nanocomposite revealed several functional groups including carboxyl (C=O) and epoxy (C-O-C) groups that act as multipoint covalent binding sites between the lipase and the Fe3O4/cellulose nanocomposite and consequently increasing lipase immobility and stability. The immobilized lipase showed a high thermo-stability as it retained about 70% of its activity at 80 °C after 30 min. The kinetics of immobilized lipase revealed that the Km and Vmax values were 0.02 mM and 32.47 U/mg protein, respectively. Moreover, the immobilized lipase showed high stability and reusability for transesterification/esterification reactions for up to four cycles with a slight decline in the enzyme activity. Furthermore, the produced biodiesel characteristics were compatible with the standards, indicating that the biodiesel obtained is doable and may be utilized in our daily life as a diesel fuel.

Unraveling Structural Carboxyl Defects in g‐C3N4 for Improved Photocatalytic H2 Evolution via Alternating Hydrogen‐Oxygen‐Plasma Treatment

AbstractStructural defect‐endowed photocatalysts are being increasingly recognized due to the enhanced catalytic activity of multiple defect sites (e.g., vacancies or functional groups). However, because of the excessive destruction effect of conventional chemical oxidation methods toward carboxyl defects engineering, the mechanism is still unclear and practice is rare in developing high‐quality structural carboxyl defect‐involved g‐C3N4. Herein, an alternating hydrogen‐oxygen‐plasma treatment is proposed to endow the g‐C3N4 with enriched vacancy defect sites for the subsequent immobilization of carboxyl groups, thus overcoming the problem of lacking of covalent binding sites in g‐C3N4 in developing carboxyl defective g‐C3N4 photocatalysts. The alternating hydrogen‐oxygen‐plasma treatment does not only influence the defect structure of g‐C3N4, but also changes its morphology, optimizes the electronic distribution, and increases the separation efficiency of photogenerated electrons and holes, thereby increasing photocatalytic H2 evolution by 7.91 times. Density functional calculations and electrochemical characterization suggest that the carboxyl defects generated by the fast H2‐O2 plasma modification lead to a local asymmetric electron environment, which enhances carrier separation capability and significantly improves H2 generation activity. This study provides a new insight into the rational design and fabrication of defect‐containing photocatalysts, carbon materials, and polymers.

Structure-Guided Discovery of the Novel Covalent Allosteric Site and Covalent Inhibitors of Fructose-1,6-Bisphosphate Aldolase to Overcome the Azole Resistance of Candidiasis.

Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis, liquid chromatography-mass spectrometry, and the crystallographic structures of APO-CaFBA, CaFBA-G3P, and C157S-2a4 revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant C. albicans, and compound 2a11 can inhibit the growth of azole-resistant strains 103 with the MIC80 of 1 μg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.