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Studies have highlighted the great potential of Oudemansiella canarii laccase in degrading synthetic dyes for reducing their toxicity. Immobilization of enzymes improves usability in degradation processes and the present work succeeded in immobilizing this laccase onto MANAE-agarose. Immobilization improved pH, thermal, and storage stabilities. Both, free and immobilized enzymes presented Michaelis-Menten kinetics with the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with K m values of 0.056 ± 0.003 and 0.195 ± 0.022 mM, respectively. Immobilization increased V max 1.27-fold. NaCl caused incomplete (hyperbolic) inhibition, which was satisfactorily described by the one-substrate one-modifier mechanism. Immobilization reduced the maximal inhibition by NaCl from 80.2 to 55.7 %. The effect of Na 2 SO 4 was predominantly stimulation, but inhibition of the free enzyme occurred at high substrate concentrations. Stimulation of the immobilized enzyme by Na 2 SO 4 was much more pronounced. It strongly depended on the substrate concentration and was much stronger (up to 300 %) at low substrate concentrations. The combined effects of substrate and sulfate on the immobilized laccase could be satisfactorily described by the one-substrate one-modifier mechanism. The modified response of the immobilized O. canarii laccase to NaCl and Na 2 SO 4 considerably favors its use as a tool in bioremediation processes because environmental contamination by salts frequently represents a strong operational challenge., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Canavan disease is caused by mutations in the ASPA gene, leading to diminished catalytic activity of aspartoacylase in the brain. Clinical missense mutations are found throughout the enzyme structure, with many of these mutated enzymes having not only decreased activity but also compromised stability. High-throughput screening of a small molecule library has identified several compounds that significantly increase the thermal stability of the E285A mutant enzyme, the most predominant clinical mutation in Canavan disease, while having a negligible effect on the native enzyme. Based on the initial successes, some structural analogs of these initial hits were selected for further examination. Glutathione, NAAG and patulin were each confirmed to be competitive inhibitors, indicating the binding of these compounds at the dimer interface or near the active site of the E285A enzyme. The experimental results were theoretically examined with the help of the docking analysis method. The structure activity-guided optimization of these compounds can potentially lead to potential pharmacological chaperones that could alleviate the detrimental effect of ASPA mutations in Canavan patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pesticides, including fungicides, are one of the important groups of environmental toxins that affect human and animal health. Studies have shown that these compounds are considered chemical pollutants. Carbendazim is a systemic fungicide. Unfortunately, excessive use of carbendazim has caused environmental pollution all over the world. In this study, the effect of carbendazim on the enzyme elastase (secreted from the endocrine gland of the pancreas) has been investigated. In a study, the performance and reaction of carbendazim with elastase were investigated using spectroscopic techniques. The stability and structure of elastase enzymes were studied under the influence of carbendazim. The results of fluorescence emission and UV-visible absorption spectrum showed that in the presence of carbendazim, there is an increase in UV-Vis absorption and a decrease in the intensity of the intrinsic fluorescence emission in the protein spectrum. Additionally, a decrease in the thermal stability of elastase was observed in the presence of carbendazim. The stability and structure of elastase enzyme were investigated in the presence of carbendazim. The results revealed that the UV-Vis absorption increased due to the presence of carbendazim, as indicated by the hyperchromic spectrum at 220 and 280 nm peaks. Additionally, the intrinsic fluorescence emission in the protein spectrum decreased with increasing carbendazim concentration at three different temperatures (298, 303, and 313 K). Moreover, the study demonstrated that the TM decreased from 2.59 to 4.58 with the increase of carbendazim, suggesting a decrease in the stability of the elastase structure in response to the elevated carbendazim concentration. According to the results of the research, the interaction between elastase and carbendazim has occurred, and changes have been made in the enzyme under the influence of carbendazim. The formation of the complex between elastase and carbendazim was consistent with the results obtained from molecular simulation and confirmed the thermodynamic data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Enzymatic degradation of alginate for the preparation of alginate oligosaccharides (AOS) is currently receiving significant attention in the field. AOS has been shown to promote crop growth and improve plant resistance to abiotic stresses. In this study, two PL6 family alginate lyases, AlyRmA and AlyRmB, were expressed and characterized. These enzymes demonstrate exceptional activity and stable thermophilicity compared to other known alginate lyases. AlyRmA (8855.34 U/mg) and AlyRmB (7879.44 U/mg) exhibited excellent degradation activity towards sodium alginate even at high temperatures (70 °C). The AlyRmA and AlyRmB were characterized and utilized to efficiently produce AOS. The study investigated the promotional effect of AOS on the growth of Brassica napus L. seedlings in a saline-alkaline environment. The results of this study demonstrate the high activity and thermal stability of AlyRmA and AlyRmB, highlighting their potential in the preparation of AOS. Moreover, the application of AOS prepared by AlyRmB could enhance the resistance of Brassica napus L. to saline-alkali environments, thereby broadening the potential applications of AOS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops., (© 2021. The Author(s).)

The oxygenases have attracted considerable attention in enzyme-mediated bioremediation of xenobiotic compounds due to their high specificity, cost-effectiveness, and targeted field applications. Here, we performed a functional metagenomics approach to cope with culturability limitations to isolate a novel extradiol dioxygenase. Fosmid clone harboring dioxygenase gene was sequenced and analyzed by bioinformatics tools. One ring-cleaving dioxygenase RW4-MPC (metapyrocatechase) was purified and characterized to examine its degradation efficiency. The RW4-MPC was significantly active in the temperature and pH range of 5 to 40 °C, and 7-10, respectively, with an optimum temperature of 25 °C and pH 8. To gain insight into observed differential activity, Small-Angle X-ray Scattering (SAXS) data of the protein samples were analyzed, which brought forth that the RW4-MPC molecules form tight globular tetramers in solution. This native association was stable till 35 °C, and protein started to associate at higher temperatures, explaining heat-induced loss of function. Similarly, RW4-MPC aggregated or lost globular profile below pH 7 or at pH 10, respectively. The kinetic parameters showed the six folds high catalytic efficiency of RW4-MPC towards 2,3-dihydroxy biphenyl than catechol and its derivatives. RW4-MPC molecules showed remarkable retention of functionality in hypersaline conditions with more than 70% activity in a buffer having 3 M NaCl concentration. In concordance, SAXS data analysis showed retention of functional shape profile in hypersaline conditions. The halotolerant and oxygen insensitive nature of this enzyme makes it a potential candidate for bioremediation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCF FBXO9 , that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCF FBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCF FBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness., (© 2021. The Author(s).)

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPI E105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPI E105D-GFP ). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

Protein aggregation, such as amyloid fibril formation, is molecular hallmark of many neurodegenerative disorders including Alzheimer's, Parkinson's, and Prion disease. Indole alkaloids are well-known as the compounds having the ability to inhibit protein fibrillation. In this study, we experimentally and computationally have investigated the anti-amyloid property of a derivative of a synthesized tetracyclic indole alkaloid (TCIA), possessing capable functional groups. The fibrillation reaction of Hen White Egg Lysozyme (HEWL) was performed in absence and presence of the indole alkaloid. For quantitative analysis, we used Thioflovin T binding assay which showed ~50% reduction in fibril formation in the presence of 20 μM TCIA. Using TEM imaging, we observed a significant morphological change in our model protein in the presence of TCIA. In addition, we exploited FT-IR assay by which Amide I peak's shifting toward lower wavenumber was clearly observed. Using Molecular Docking, the interaction of the inhibitor (TCIA) with the protein's amyloidogenic region was modeled. Also, different biophysical parameters were calculated by Molecular Dynamics (MD) simulation. Various biochemical assays, conformational change, and hydrophobicity exposure of the protein during amyloid formation indicated that the compound assists HEWL to keep its native structure via destabilizing β-sheet structure., (Copyright © 2021. Published by Elsevier B.V.)

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504 T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a T m value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

As an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4-30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5-35 °C and pH 6.0-12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low K m value of 1.0 mg/ml and V max 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

The dimeric dihydropyrimidine dehydrogenase (DPD), metalloenzyme, an adjunct anti-cancer drug target, contains highly specialized 4 × Fe 2+ 4 S 2- 4 clusters per chain. These clusters facilitate the catalysis of the rate-limiting step in the pyrimidine degradation pathway through a harmonized electron transfer cascade that triggers a redox catabolic reaction. In the process, the bulk of the administered 5-fluorouracil (5-FU) cancer drug is inactivated, while a small proportion is activated to nucleic acid antimetabolites. The occurrence of missense mutations in DPD protein within the general population, including those of African descent, has adverse toxicity effects due to altered 5-FU metabolism. Thus, deciphering mutation effects on protein structure and function is vital, especially for precision medicine purposes. We previously proposed combining molecular dynamics (MD) and dynamic residue network (DRN) analysis to decipher the molecular mechanisms of missense mutations in other proteins. However, the presence of Fe 2+ 4 S 2- 4 clusters in DPD poses a challenge for such in silico studies. The existing AMBER force field parameters cannot accurately describe the Fe 2+ center coordination exhibited by this enzyme. Therefore, this study aimed to derive AMBER force field parameters for DPD enzyme Fe 2+ centers, using the original Seminario method and the collation features Visual Force Field Derivation Toolkit as a supportive approach. All-atom MD simulations were performed to validate the results. Both approaches generated similar force field parameters, which accurately described the human DPD protein Fe 2+ 4 S 2- 4 cluster architecture. This information is crucial and opens new avenues for in silico cancer pharmacogenomics and drug discovery related research on 5-FU drug efficacy and toxicity issues.

Isocitrate dehydrogenase 1 (IDH1) mutant R132H, promoting the oncometabolite D-2-hydroxyglutarate (D2HG), is a driver mutation and an emerging therapeutic target in glioma. This study identified a novel mutant IDH1 inhibitor, WM17, by virtual screening and enzymatic confirmation. It could bind to and increase mutant IDH1 protein's thermostability in both endogenous heterozygous cells and exogenous overexpressed cells. Consequently, WM17 reversed the accumulation of D2HG and histone hypermethylation in IDH1 mutated cells. Finally, we concluded that WM17 significantly inhibited cell migration in IDH1 mutated glioma cells, although it has no apparent effect on cell proliferation. Further studies are guaranteed toward the development of WM17 as a therapeutic agent for IDH1 mutated glioma., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Garlic is a well-known example of natural self-defence system consisting of an inactive substrate (alliin) and enzyme (alliinase) which, when combined, produce highly antimicrobial allicin. Increase of alliinase stability and its activity are of paramount importance in various applications relying on its use for in-situ synthesis of allicin or its analogues, e.g., pulmonary drug delivery, treatment of superficial injuries, or urease inhibitors in fertilizers. Here, we discuss the effect of temperature, pH, buffers, salts, and additives, i.e. antioxidants, chelating agents, reducing agents and cosolvents, on the stability and the activity of alliinase extracted from garlic. The effects of the storage temperature and relative humidity on the stability of lyophilized alliinase was demonstrated. A combination of the short half-life, high reactivity and non-specificity to particular proteins are reasons most bacteria cannot deal with allicin's mode of action and develop effective defence mechanism, which could be the key to sustainable drug design addressing serious problems with escalating emergence of multidrug-resistant (MDR) bacterial strains., Competing Interests: The authors have declared that no competing interests exist.

T-cell and B-cell acute lymphoblastic leukemias (T-ALL, B-ALL) are aggressive hematological malignancies characterized by an accumulation of immature T- or B-cells. Although patient outcomes have improved, novel targeted therapies are needed to reduce the intensity of chemotherapy and improve the prognosis of high-risk patients. Using cell lines, primary cells and patient-derived xenograft (PDX) models, we demonstrate that ALL cells viability is sensitive to NVP-BEP800, an ATP-competitive inhibitor of Heat shock protein 90 (HSP90). Furthermore, we reveal that lymphocyte-specific SRC family kinases (SFK) are important clients of the HSP90 chaperone in ALL. When PDX mice are treated with NVP-BEP800, we found that there is a decrease in ALL progression. Together, these results demonstrate that the chaperoning of SFK by HSP90 is involved in the growth of ALL. These novel findings provide an alternative approach to target SRC kinases and could be used for the development of new treatment strategies for ALL.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor β (TGF-β) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme K m and V max values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme's co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

This paper aims to investigate the effects of some salts (NaCl, (NH 4 ) 2 SO 4 and Na 2 SO 4 ) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH 4 ) 2 SO 4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram's staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS-PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10-90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with K M and V max of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H 2 O 2 ), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens . The structure of this protease and its highest-priority substrate β-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein-protein (protease from HM48 and β-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli . We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. K m values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH 2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH 2 nanospheres showed high loading capacity of 103.8 μg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications., Competing Interests: Declaration of competing interest The authors have no potential conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Herein acid phosphatase isoenzyme was extracted from the C. murale seedlings. The purification was accomplished by chromatographic techniques and passing through DEAE-cellulose and Sephadex G-100 column. The specific activity of acid phosphatase 5.75 U/mg of protein was obtained with 66 purification fold 15.8% yield and molecular mass was 29 kDa with very faint bands corresponding to 18 kDa and 14 kDa. The maximal activity at pH 5.0 and 50 °C best illustrated by first order kinetics. When temperature was raised (55 °C to 75 °C), the deactivation rate constant was increased from 0.001 to 0.014 min -1 , while half-life was decreased from 693 to 49 min -1 . The results of activity collected at different temperature were then used to estimate, activation energy of hydrolysis reaction (Ea = 47.59 kJmol -1 ). A high Z-value (18.86 °C min -1 ) was obtained indicating a less sensitivity towards temperatures. The residual activity examinations were carried out from 55 °C to 75 °C and assessing the Deactivation Energy (Ed 116.39 kJmol -1 ), Enthalpy change (ΔH° 113.55kJmol -1 ), Entropy change (ΔS° 110.33kJmol -1 ) and change in Gibbs free energy (ΔG° 10.02 kJmol -1 ). Taken together, thermodynamic parameters confirm the high stability of enzyme and show potential commercial applicability., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metabolic rewiring is a mechanism of adaptation to unfavorable environmental conditions and tumor progression. TRAP1 is an HSP90 molecular chaperone upregulated in human colorectal carcinomas (CRCs) and responsible for downregulation of oxidative phosphorylation (OXPHOS) and adaptation to metabolic stress. The mechanism by which TRAP1 regulates glycolytic metabolism and the relevance of this regulation in resistance to EGFR inhibitors were investigated in patient-derived CRC spheres, human CRC cells, samples, and patients. A linear correlation was observed between TRAP1 levels and 18 F-fluoro-2-deoxy-glucose ( 18 F-FDG) uptake upon PET scan or GLUT1 expression in human CRCs. Consistently, TRAP1 enhances GLUT1 expression, glucose uptake, and lactate production and downregulates OXPHOS in CRC patient-derived spheroids and cell lines. Mechanistically, TRAP1 maximizes lactate production to balance low OXPHOS through the regulation of the glycolytic enzyme phosphofructokinase-1 (PFK1); this depends on the interaction between TRAP1 and PFK1, which favors PFK1 glycolytic activity and prevents its ubiquitination/degradation. By contrast, TRAP1/PFK1 interaction is lost in conditions of enhanced OXPHOS, which results in loss of TRAP1 regulation of PFK1 activity and lactate production. Notably, TRAP1 regulation of glycolysis is involved in resistance of RAS-wild-type CRCs to EGFR monoclonals. Indeed, either TRAP1 upregulation or high glycolytic metabolism impairs cetuximab activity in vitro, whereas TRAP1 targeting and/or inhibition of glycolytic pathway enhances cell response to cetuximab. Finally, a linear correlation between 18 F-FDG PET uptake and poor response to cetuximab in first-line therapy in human metastatic CRCs was observed. These results suggest that TRAP1 is a key determinant of CRC metabolic rewiring and favors resistance to EGFR inhibitors through regulation of glycolytic metabolism., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system 1-6 . The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS 7-17 , and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

Lecithin:cholesterol acyltransferase (LCAT) is a unique plasma enzyme able to esterify cholesterol, and it plays an important role in HDL maturation and promotion of reverse cholesterol transport. Familial LCAT deficiency (FLD; OMIM number 245900) is a rare recessive disease that results from loss-of-function mutations in the LCAT gene and has no cure. In this study, we assessed the in vitro efficacy of a novel small-molecule LCAT activator. Cholesterol esterification rate (CER) and LCAT activity were tested in plasma from six controls and five FLD homozygous carriers of various LCAT mutations at different doses of the compound (0.1, 1, and 10 µg/ml). In control plasma, the compound significantly increased both CER ( P < 0.001) and LCAT activity ( P = 0.007) in a dose-dependent manner. Both CER and LCAT activity increased by 4- to 5-fold, reaching maximum activation at the dose of 1 µg/ml. Interestingly, Daiichi Sankyo compound produced an increase in CER in two of the five tested LCAT mutants (Leu372--Arg and Val309--Met), while LCAT activity increased in three LCAT mutants (Arg147--Trp, Thr274--Ile and Leu372--Arg); mutant Pro254--Ser was not activated at any of the tested doses. The present findings form the basis for personalized therapeutic interventions in FLD carriers and support the potential LCAT activation in secondary LCAT defects. SIGNIFICANCE STATEMENT: We characterized the pharmacology of a novel small-molecule LCAT activator in vitro on a subset of naturally occurring LCAT mutants. Our findings form the basis for personalized therapeutic interventions for familial LCAT deficiency carriers, who can face severe complications and for whom no cure exists., Competing Interests: K.K., K. Y., and N. K. are employees of Daiichi Sankyo., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

A β-glucosidase gene (bsbgl1a) from Bacillus sp. CGMCC 1.16541 was expressed in Escherichia coli BL21 and subsequently characterized. The amino acid sequence shared 83.64% identity with β-glucosidase (WP&#95;066390903.1) from Fictibacillus phosphorivorans. The recombinant β-glucosidase (BsBgl1A) had a molecular weight of 52.2 kDa and could hydrolyze cellobiose, cellotriose, cellotetrose, p-nitrophenyl-β-D-glucopyranoside (pNPG), and p-nitrophenyl-β-D-xylopyranoside (pNPX). Optimal activity for BsBgl1A was recorded at 45 °C with a pH between 5.6 and 7.6, and 100% of its activity was maintained after a 24 h incubation between pH 4 and 9. Kinetic characterization revealed an enzymatic turnover (Kcat) of 616 ± 2 s -1 (with cellobiose) and 3.5 ± 0.1 s -1 (with p-nitrophenyl-β-D-glucopyranoside). Interestingly, the recombinant enzyme showed cupric ion (Cu 2+ ), sodium dodecyl sulfate (SDS) and alcohol tolerance at 10 mM for Cu 2+ and 10% for both SDS and alcohol. Additionally, BsBgl1A had high tolerance for glucose (Ki = 2095 mM), which is an extremely desirable feature for industrial applications. Following the addition of BsBgl1A (0.05 mg/ml) to a commercial cellulase reaction system, glucose yields from sugarcane bagasse increased 100% after 1 day at 45 °C. This work identifies a Cu 2+ , SDS, alcohol, and glucose tolerant GH1 β-glucosidase with potential applications in the hydrolysis of cellulose for the bioenergy industry.

The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive ( e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ray et al.)

The sterile α motif, also called the SAM domain, is known to form homo or heterocomplexes that modulate diverse biological functions through the regulation of specific protein-protein interactions. The MAPK pathway of budding yeast Saccharomyces cerevisiae is comprised of a three-tier kinase system akin to mammals. The MAPKKK Ste11 protein of yeast contains a homodimer SAM domain, which is critical for transmitting cues to the downstream kinases. The structural stability of the dimeric Ste11 SAM is maintained by hydrophobic and ionic interactions at the interfacial amino acids. The urea-induced equilibrium-unfolding process of the Ste11 SAM domain is cooperative without evidence of any intermediate states. The native-state H/D exchange under subdenaturing conditions is a useful method for the detection of intermediate states of proteins. In the present study, we investigated the effect of ionic strength on the conformational stability of the dimer using the H/D exchange experiments. The hydrogen exchange behavior of the Ste11 dimer under physiological salt concentrations reveals two partially unfolded metastable intermediate states, which may be generated by a sequential and cooperative unfolding of the five helices present in the domain. These intermediates appear to be significant for the reversible unfolding kinetics via hydrophobic collapse. In contrast, higher ionic concentrations eliminate this cooperative interactions that stabilize the pairs of helices.

The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such entrainment did not seem to perturb enzyme function, but it did significantly enhance enzyme stability against several denaturing stimuli such as heat, organic solvents, and chaotropic agents. This improvement in performance was found to be general and independent of the number of independent subunits required and of the number of catalytically active enzymes packaged. Packaged enzymes were found by measurements of intrinsic tryptophan fluorescence to retain some of their native folded structure even longer than their catalytic activity, suggesting that protein folding is a significant component of the observed catalytic benefits. While we are unable to distinguish between kinetic and thermodynamic effects - including inhibition of enzyme unfolding, acceleration of refolding, and biasing of folding equilibria - VLP packaging appears to represent a useful general strategy for the stabilization of enzymes that operate on diffusible substrates and products.

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from white rot fungi Phanerochaete chrysosporium can be used for constructing biosensors and biofuel cells, for bleaching cotton in textile industry, and recently, the enzyme has found an important application in biomedicine as an antimicrobial and antibiofilm agent. Stability and activity of the wild-type (wt) CDH and mutants at methionine residues in the presence of hydrogen peroxide were investigated. Saturation mutagenesis libraries were made at the only methionine in heme domain M65 and two methionines M685 and M738 in the flavin domain that were closest to the active site. After screening the libraries, three mutants with increased activity and stability in the presence of peroxide were found, M65F with 70% of residual activity after 6 h of incubation in 0.3 M hydrogen peroxide, M738S with 80% of residual activity and M685Y with over 90% of residual activity compared to wild-type CDH that retained 40% of original activity. Combined mutants showed no activity. The most stable mutant M685Y with 5.8 times increased half-life in the presence of peroxide showed also 2.5 times increased k cat for lactose compared to wtCDH and could be good candidate for applications in biofuel cells and biocatalysis for lactobionic acid production.

Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%-40% ethanol solutions. The optimum temperature of CPUTNase was 43°C. This enzyme showed urethanase activity at pH 5.5-10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) enables the investigation of protein folding in solution. Herein, a proof-of-concept for obtaining structural information about the folding of a protein in dependency of the amount of an organic cosolvent in the aqueous medium by means of this IMS-MS method is presented. By analyzing the protein with native nano-electrospray ionization IMS-MS, the impact of acetonitrile as a representative organic cosolvent and/or pH values on the folding of an enzyme was successfully evaluated in a fast and straightforward fashion, as exemplified for an ene reductase from Gluconobacter oxydans. The IMS-MS results are in agreement with findings from the nicotinamide adenine dinucleotide phosphate (NADPH)-based spectrophotometric enzyme activity tests under analogous conditions, and thus, also rationalizing these "wet" analytical data. For this ene reductase, a higher tolerance against CH 3 CN in the presence of a buffer was observed by both analytical methods. The results suggest that this IMS-MS methodology could be a useful complementary tool to existing methods in process optimization and fine-tuning of solvent conditions for biotransformations., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02 (GtGBE, EC 2.4.1.18) does not possess the thermostability required by modified starch industry. To increase its thermostability, a rational design strategy was used to introduce additional salt bridges into GtGBE. The strategy involved in mutation of individual residues to form "local" two-residue salt bridges. Accordingly, five of local salt bridges (Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and I571D-R569 mutants) were separately introduced into GtGBE. The half-times of these mutants at 60 °C were 17% to 51% longer than that of wild-type. Subsequently, these two-residue salt bridges were extended to form salt bridge networks (Q231R/K-D227-D131H, T339D/E-K335-I291H, and I571D-R569-R617H mutants). Among these mutants, except I571D-R569-R617H, the half-times of Q231R/K-D227-D131H, T339D/E-K335-I291H mutants at 60 °C were 15%, 17%, 21% and 17% longer than those of the corresponding two-residue salt bridges, respectively. The results showed that design and introduction of salt bridges improves enzyme thermostability in GtGBE., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The stability of Debaryomyces nepalensis NCYC 3413 xylose reductase, a homodimeric enzyme recombinantly expressed and purified from E. coli Rosetta cells, was studied at different pH ranging from 5.0 to 10.0. Deactivation kinetics at different pH were studied by analyzing residual activity of the recombinant enzyme over time at 40 °C whereas conformational changes and stability dependence were investigated by using circular dichroism and differential scanning calorimetry. Four osmolytes viz. glycerol, sucrose, trehalose and sorbitol were explored for their effect on the deactivation and melting temperatures of the enzyme under neutral and extreme pH conditions. The enzyme was found to be catalytically and structurally stable at pH 7.0 with half-life of 250 min and a melting temperature of 50 °C. It was found that alteration in both secondary and tertiary structures caused enzyme deactivation in acidic pH while increased deactivation rates at alkaline pH was attributed to the variation of tertiary structure over time. Estimated thermodynamic parameters also showed that the enzyme stability was highest at neutral pH with ΔH of 348 kcal/mole and ΔG 40 of 9.53 kcal/mole. All four osmolytes were effective in enhancing enzyme stability by several folds at extreme pH with sorbitol being the most efficient, which increased enzyme half-life by 11-fold at pH 10.0 and 8-fold at pH 5.0.

Ethylene is an important plant hormone that controls growth, development, aging and stress responses. The rate-limiting enzymes in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylate synthases (ACSs), are strictly regulated at many levels, including posttranslational control of protein half-life. Reversible phosphorylation/dephosphorylation events play a pivotal role as signals for ubiquitin-dependent degradation. We showed previously that ABI1, a group A protein phosphatase type 2C (PP2C) and a key negative regulator of abscisic acid signaling regulates type I ACS stability. Here we provide evidence that ABI1 also contributes to the regulation of ethylene biosynthesis via ACS7, a type III ACS without known regulatory domains. Using various approaches, we show that ACS7 interacts with ABI1, ABI2 and HAB1. We use molecular modeling to predict the amino acid residues involved in ABI1/ACS7 complex formation and confirm these predictions by mcBiFC-FRET-FLIM analysis. Using a cell-free degradation assay, we show that proteasomal degradation of ACS7 is delayed in protein extracts prepared from PP2C type A knockout plants, compared to a wild-type extract. This study therefore shows that ACS7 undergoes complex regulation governed by ABI1, ABI2 and HAB1. Furthermore, this suggests that ACS7, together with PP2Cs, plays an essential role in maintaining appropriate levels of ethylene in Arabidopsis.

RAF kinases (ARAF, BRAF, and CRAF) are highly conserved enzymes that trigger the RAF-MEK1/2-ERK1/2 (MAPK) pathway upon activation of RAS. Despite enormous clinical interest, relatively little is known on the role of RAFs in mediating immune responses. Here, we investigated the role of RAF kinases and MEK1/2 in dendritic cells (DCs), the central regulators of T cell-mediated antitumor immune responses and the adaptive immune system. We demonstrate that RAF kinases are active and stabilized at their protein levels during DC differentiation. Inhibition of RAF kinases but not MEK1/2 impaired the activation of DCs in both mice and human. As expected, DCs treated with RAF inhibitors show defects in activating T cells. Further, RAF and MEK1/2 kinases are directly required for the activation and proliferation of CD4 + T cells. Our observations suggest that RAF and MEK1/2 have independent roles in regulating DC function that has important implications for administering RAF-MAPK inhibitors in the clinics.

Thermal shift assay is a fluorescence dye based biochemical method to determine the melting point of a protein. It can be used to investigate the ligand-induced stabilization of proteins and helps to increase the likelihood of crystallization in biological samples. Dimeric proteins like soluble guanylyl cyclase (sGC) have specific structural and functional properties which may pose a challenge in thermal shift measurements. In this paper, thermal shift assay was used to examine ligand-induced thermostabilization of the dimeric heme-containing protein soluble guanylyl cyclase. Adjustment of the parameters buffer solution, pH, protein / dye ratio and protein amount per well yielded a one-phase melting curve of sGC with a sharp transition and high reproducibility. We found that thermal shift measurement is not affected by heme state or heme content of the enzyme preparation. We used the method to investigate the thermostabilization of sGC induced by the heme-mimetic activator drugs cinaciguat, BAY 60-2770 and BR 11257 in combination with non-hydrolyzable nucleotides. Measurements with the dicarboxylic drugs cinaciguat and BAY 60-2770 yielded steep melting curves with high amplitudes. In contrast, in the presence of the monocarboxylic sGC activator BR 11257, melting curves appear flattened in the dye-based measurements. In the present paper, we show that activity-based thermostability measurements are superior to dye-based measurements in detecting the thermostabilizing influence of sGC activator drugs., Competing Interests: Declaration of Competing Interest PS is a full-time employee of Bayer AG., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Thioamide substitutions in peptides can be used as fluorescence quenchers in protease sensors and as stabilizing modifications of hormone analogs. To guide these applications in the context of serine proteases, we here examine the cleavage of several model substrates, scanning a thioamide between the P3 and P3' positions, and identify perturbing positions for thioamide substitution. While all serine proteases tested were affected by P1 thioamidation, certain proteases were also significantly affected by other thioamide positions. We demonstrate how these findings can be applied by harnessing the combined P3/P1 effect of a single thioamide on kallikrein proteolysis to protect two key positions in a neuropeptide Y-based imaging probe, increasing its serum half-life to >24 h while maintaining potency for binding to Y1 receptor expressing cells. Such stabilized peptide probes could find application in imaging cell populations in animal models or even in clinical applications such as fluorescence-guided surgery.

Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC 50 values were found to be in a range of 1-3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA-PEtOx 30 -IDA (PEtOx: poly(2-ethyl-2-oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx-IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Sphingosine kinase 1 (SphK1) is a lipid kinase which plays vital role in the regulation of varieties of biological processes including, cell growth, apoptosis and mitogenesis. In the present study, we investigated the guanidinium chloride (GdmCl)-induced denaturation of SphK1 at pH 8.0 and 25 °C using two different spectroscopic probes, i.e., mean residue ellipticity at 222 nm ([θ] 222 ) and fluorescence emission maxima (λ max ). A significant overlap between the transition curves obtained from both the spectral properties indicate that GdmCl-induced unfolding of SphK1 follows two-state process i.e., Native (N) ⇌ Denatured (D) state. Interestingly, a visible protein aggregation was observed at low concentrations of GdmCl ([GdmCl] ≤ 1.5 M). The analysis of transition curves was done to estimate the thermodynamic parameters associated with the stability of SphK1. To complement our experimental findings, 100 ns molecular dynamics (MD) simulations were performed. Spectroscopic studies together with MD simulations provided mechanistic insights of unfolding pathway of SphK1 along with its stability parameters., Competing Interests: Declaration of competing interest Author declares no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l-asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l-asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 μmol min - 1  mg - 1 towards l-asparagine. The enzyme exhibited a 10% activity towards d-asparagine as compared to 100% against l-asparagine. No detectable activity was observed towards l- or d-glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent K m and V max values of 3.1 mM and 833 μmol min - 1  mg - 1 , respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol - 1 . To the best of our knowledge, this is the first characterization of a plant-type l-asparaginase from class Thermococci of phylum Euryarchaeota., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mostly, enzyme activity is reduced after immobilization of enzyme within MOF due to unfavourable conformational changes occurred during the immobilization procedure. In this context, lipase was activated by surfactants (in order to get a highly active enzyme) followed by encapsulation within zeolitic imidazolate framework (ZIF)-8 via one-pot facile self-assembly method. The prepared lipase-SDS ZIF-8 exhibited 250% enhanced activity compared to native form. The prepared biocomposite was characterized and analysed by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and scanning electron microscopy (SEM). Thermo-stability was determined for prepared lipase-SDS ZIF-8 biocomposite in the range of 50-70 °C, which showed more than two-folds enhanced stability in terms of half-life. Further, immobilized lipase retained 76% of residual activity even after six repetitive cycles and, it showed 91% residual activity after twenty days of long term storage. Finally, lipase-SDS ZIF-8 was tested for chemical stability in polar denaturing solvents which showed excellent stability as compared to free lipase., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes two uracil DNA glycosylases (UDGs): Tba UDG247 and Tba UDG194. Herein, we characterized biochemically Tba UDG194. Compared with Tba UDG247, Tba UDG194 exhibits different biochemical characteristics. At >85 °C, >90 cleavage percentage was observed, suggesting that Tba UDG194 can remove uracil from DNA at physiological temperature of its host. Thus, the enzyme is the most thermophilic glycosylase among all the reported UDGs. Furthermore, the optimal pH of the enzyme activity was estimated to be 10, which is higher than that of Tba UDG247. Similar to Tba UDG247, Tba UDG194 activity is independent on a divalent metal ion. Mn 2+ , Zn 2+ and Cu 2+ display inhibitory effect on the enzyme activity at varied degreed whereas Mg 2+ and Ca 2+ have no detectable effect on the enzyme activity. In addition, Tba UDG194 is a salt-tolerant enzyme that retains compromised activity at 600 mM NaCl. Furthermore, Tba UDG 194 displays the following substrate preference: U ≈ U/G > U/T > U/A > U/C. The Arrhenius activation energy was estimated to be 20.1 ± 3.4 kcal/mol, theoretically representing the energy barrier for uracil removal from DNA by Tba UDG194. Overall, our observations suggest that Tba UDG194 might be involved in removal of uracil in DNA in Thermococcus cells., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors., (Copyright © 2019. Published by Elsevier B.V.)

A 61.3 kDa Phenol hydroxylase (PheA) was purified and characterized from Pseudomonas sp. KZNSA (PKZNSA). Cell free extract of the isolate grown in mineral salt medium supplemented with 600 ppm phenol showed 21.58 U/mL of PheA activity with a specific activity of 7.67 U/mg of protein. The enzyme was purified to 1.6-fold with a total yield of 33.6%. The purified PheA was optimally active at pH 8 and temperature 30 °C, with ≈95% stability at pH 7.5 and temperature 30 °C after 2 h. The Lineweaver-Burk plot showed the v max and K m values of 4.04 µM/min and 4.03 µM, respectively, for the substrate phenol. The ES-MS data generated from the tryptic digested fragments of pure protein and PCR amplification of a ≈600 bp gene from genomic DNA of PKZNSA lead to the determination of complete amino acid and nucleotide sequence of PheA. Bioinformatics tools and homology modelling studies indicated that PheA from PKZNSA is likely a probable protein kinase UbiB (2-octaprenylphenol hydroxylase) involving Lys and Asp at positions 153 and 288 for binding and active site, respectively. Characterization and optimization of PheA activity may be useful for a better understanding of 2,4-dichlorophenol degradation by this organism and for potential industrial application of the enzyme., Competing Interests: Declaration of Competing Interest All the authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Improving enzyme stability in the presence of organic solvent is crucial for non-aqueous catalysis. In this study, directed evolution was applied to improve the tolerance of metalloprotease PT121 towards organic solvent. In presence of acetonitrile and acetone, three mutants (T46Y, H224 F, and H224Y) of PT121 showed excellent solvent stability, which increased their half-lives by 1.2-3.5-fold as compared to the wild-type enzyme. Kinetic constants (K M and k cat values) of the caseinolysis reaction presented H224 F and H224Y mutants have higher affinity than the wild-type, but T46Y mutant were similar to those of the wild-type enzyme. Interestingly, combined mutants T46Y/H224 F and T46Y/H224Y mutants presented awesome stability and excellent caseinolytic activity. Molecular dynamic simulation suggest that improved enzyme stability may be attributed to extensive non-covalent bond network resulting in a more compact structure. Disruption of the disulphide bond formation between Cys-30 and Cys-58 residues in the F56 V mutant is possibly the reason behind its low stability among all the selected mutants. Additionally, T46Y/H224 F and T46Y/H224Y showed a higher peptide synthetic activity in the presence of organic solvents than the wild-type, which renders these mutant enzymes as promising biocatalysts for biotechnological applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Mimicking the cellular environment, metal-organic frameworks (MOFs) are promising for encapsulating enzymes for general applications in environments often unfavorable for native enzymes. Markedly different from previous researches based on bulk solution synthesis, here, we report the synthesis of enzyme-embedded MOFs in a microfluidic laminar flow. The continuously changed concentrations of MOF precursors in the gradient mixing on-chip resulted in structural defects in products. This defect-generating phenomenon enables multimodal pore size distribution in MOFs and therefore allows improved access of substrates to encapsulated enzymes while maintaining the protection to the enzymes. Thus, the as-produced enzyme-MOF composites showed much higher (~one order of magnitude) biological activity than those from conventional bulk solution synthesis. This work suggests that while microfluidic flow synthesis is currently underexplored, it is a promising strategy in producing highly active enzyme-MOF composites., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)