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L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC 90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site-directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent -copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the μs timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 °C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme.

Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, T m ) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOX NT1 ) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOX NT1 , 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The T m of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the T m of 65 °C for LGOX NT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

To enhance the thermal stability of Streptomyces Sp. SA-COO cholesterol oxidase, random mutagenesis was used. A random mutant library was generated using two types of error-prone PCR (single step and serial dilution) and two mutants (ChOA-M1 and ChOA-M2) with improved thermostability were obtained. The best mutant ChOA-M1 acquired three amino acid substitutions (G49T, W52K, and F62V) and improved thermostability (at 50 °C for 5 h) by 40% and increased the kcat/Km value by 23%. The optimum pH was desirably changed to encompass a broad range from alkali to acid and circular dichroism revealed no significant secondary structure changes in mutants against wild type. These findings indicated that random mutagenesis was an effective technique for optimizing cholesterol oxidase properties and make a foundation for practical applications of Cholesterol oxidase in clinical diagnosis and industrial fields., (Copyright © 2021. Published by Elsevier Inc.)

The RNA dependent RNA polymerase (RdRp) plays crucial role in virus life cycle by replicating the viral genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and acquired mutations. This study was carried out to identify mutations in RdRp as the SARS-CoV-2 spread in India. We compared 50217 RdRp sequences reported from India with the first reported RdRp sequence from Wuhan, China to identify 223 mutations acquired among Indian isolates. Our protein modelling study revealed that several mutants can potentially alter stability and flexibility of RdRp. We predicted the potential B cell epitopes contributed by RdRp and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among 223 RdRp mutants, 44% of them localises in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp to understand the impact of these mutations on SARS-CoV-2., (Copyright © 2021 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Catalytic promiscuity is the coincidental ability to catalyze nonbiological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies a potentially preadaptive residue in a promiscuous N -succinylamino acid racemase/ o -succinylbenzoate synthase (NSAR/OSBS) enzyme from Amycolatopsis sp. T-1-60. This enzyme belongs to a branch of the OSBS family which includes many catalytically promiscuous NSAR/OSBS enzymes. R266 is conserved in all members of the NSAR/OSBS subfamily. However, the homologous position is usually hydrophobic in other OSBS subfamilies, whose enzymes lack NSAR activity. The second-shell amino acid R266 is close to the catalytic acid/base K263, but it does not contact the substrate, suggesting that R266 could affect the catalytic mechanism. Mutating R266 to glutamine in Amycolatopsis NSAR/OSBS profoundly reduces NSAR activity but moderately reduces OSBS activity. This is due to a 1000-fold decrease in the rate of proton exchange between the substrate and the general acid/base catalyst K263. This mutation is less deleterious for the OSBS reaction because K263 forms a cation-π interaction with the OSBS substrate and/or the intermediate, rather than acting as a general acid/base catalyst. Together, the data explain how R266 contributes to NSAR reaction specificity and was likely an essential preadaptation for the evolution of NSAR activity.

This study reports a novel BglA9 gene of 1345 bp encoding β-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were K m (0.28 mM), V max (43.8 μmol/min/mg), k cat (38.43 s -1 ) and k cat /K m (135.5 s -1  mM -1 ). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 μmol/min/mg, 18.28 s -1 and 3.27 s -1  mM -1 for K m , V max , k cat , and k cat /K m values, respectively. The K i value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin., (Copyright © 2021. Published by Elsevier B.V.)

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The standalone metallo-β-lactamase-type thioesterase (MβL-TE), belongs to the group V nonreducing polyketide synthase agene cluster, catalyzes the rate-limiting step of product releasing. Our work first investigated on the orthologous MβL-TEs from different origins to determine which nonconserved amino acid residues are important to the hydrolysis efficiency. A series of chimeric MβL-TEs were constructed by fragment swapping and site-directed mutagenesis, in vivo enzymatic assay showed that two nonconserved residues A19 and E75 (numbering in HyTE) were critical to the catalytic performance. Protein structure modeling suggested that these two residues are located in different areas of HyTE. A19 is on the entrance to the active sites, whereas E75 resides in the linker between the two β strands which hold the metal-binding sites. Combining with computational simulations and comparative enzymatic assay, different screening criteria were set up for selecting the variants on the two noncatalytic and nonconserved key residues to improve the catalytic activity. The rational design on A19 and E75 gave five candidates in total, two (A19F and E75Q) of which were thus found significantly improved the enzymatic performance of HyTE. The double-point mutant was constructed to further improve the activity, which was increased by 28.4-fold on product accumulation comparing to the wild-type HyTE. This study provides a novel approach for engineering on nonconserved residues to optimize enzymatic performance., (© 2021 Wiley Periodicals LLC.)

Objective: To develop an endo-β-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process., Results: The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg -1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g -1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis., Conclusion: The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The group of hydrolytic enzymes synonymously known as proteases is predominantly most favored for the class of industrial enzymes. The present work focuses on the thermostable nature of these proteolytic enzymes that occur naturally among mesophilic and thermophilic microbes. The broad thermo-active feature (40-80 °C), ease of cultivation, maintenance, and bulk production are the key features associated with these enzymes. Detailing of contemporary production technologies, and controllable operational parameters including the purification strategies, are the key features that justify their industrial dominance as biocatalysts. In addition, the rigorous research inputs by protein engineering and enzyme immobilization studies add up to the thermo-catalytic features and application capabilities of these enzymes. The work summarizes key features of microbial proteases that make them numero-uno for laundry, biomaterials, waste management, food and feed, tannery, and medical as well as pharmaceutical industries. The quest for novel and/or designed and engineered thermostable protease from unexplored sources is highly stimulating and will address the ever-increasing industrial demands., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The K m values for the intrinsic as well as protein-stimulated ATPase were increased whereas the k cat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli , purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters K M and V max for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni 2+ , Cu 2+ , Mg 2+ , Zn 2+ and Ca 2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe 3+ . The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

GTP cyclohydrolase I enzyme (GTPCH-I) is a rate limiting enzyme in the biosynthesis pathway of tetrahydrobiopterin (BH4) and tetrahydrofolate (THF) compounds; latter being are an essential compounds involved in many biological functions. This enzyme has been evaluated structurally and functionally in many organisms to understand its putative role in cell processes, kinetics, regulations, drug targeting in infectious diseases, pain sensitivity in humans, and so on. In Mycobacterium tuberculosis (a human pathogen causing tuberculosis), this GTPCH-I activity has been predicted to be present in Rv3609c gene (folE) of H 37 Rv strain, which till date has not been studied in detail. In order to understand in depth, the structure and function of folE protein in M. tuberculosis H 37 Rv, in silico study was designed by using many different bioinformatics tools. Comparative and structural analysis predicts that Rv3609c gene is similar to folE protein ortholog of Listeria monocytogenes (cause food born disease), and uses zinc ion as a cofactor for its catalysis. Result shows that mutation of folE protein at 52th residue from tyrosine to glycine or variation in pH and temperature can lead to high destability in protein structure. Studies here have also predicted about the functional regions and interacting partners involved with folE protein. This study has provided clues to carry out experimentally the analysis of folE protein in mycobacteria and if found suitable will be used for drug targeting., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Deubiquitinating enzymes (DUBs) play important roles in many physiological and pathological processes by modulating the ubiquitination of their substrates. DUBs undergo post-translational modifications including ubiquitination. However, whether DUBs can reverse their own ubiquitination and regulate their own protein stability requires further investigation. To answer this question, we screened an expression library of DUBs and their enzymatic activity mutants and found that some DUBs regulated their own protein stability in an enzymatic activity- and homomeric interaction-dependent manner. Taking Ubiquitin-specific-processing protease 29 (USP29) as an example, we found that USP29 deubiquitinates itself and protects itself from proteasomal degradation. We also revealed that the N-terminal region of USP29 is critical for its protein stability. Taken together, our work demonstrates that at least some DUBs regulate their own ubiquitination and protein stability. Our findings provide novel molecular insight into the diverse regulation of DUBs., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In recent years, with the increasing public health awareness, low-calorie rare sugars have received more attention on a global scale. D-Allulose, the C-3 epimer of D-fructose, is a representative rare sugar. It displays high sweetness and excellent physiological functions, but only provides a caloric value of 0.4 kcal/g. D-Allulose 3-epimerase (DAEase) is indispensable in D-allulose production. In this study, a putative DAEase from Thermoclostridium caenicola was identified and characterized. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 °C in the presence of 1 mM Co 2+ . The half-life (t 1/2 ) at 50 °C was 13.6 h, and the melting temperature (T m ) was 62.4 °C. It was strictly metal-dependent, and the addition of Co 2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t 1/2 value at 55 °C and 4.8 °C increase in T m . Furthermore, DAEase displayed high relative activity (89.0%) at a weakly acidic pH 6.5 and produced 139.8 g/L D-allulose from 500 g/L D-fructose, achieving a conversion ratio of 28.0%. These findings suggest that T. caenicola DAEase is a promising biocatalyst for the production of D-allulose.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Glucose oxidase (Gox) is a biocatalyst that is widely applied in the food industry, as well as other biotechnological industries. However, the industrial application of Gox is hampered by its low thermostability and activity. Here, we aimed to improve the thermostability of GoxM4 from Aspergillus niger without reducing its activity due to the activity-stability trade-off. A simple and effective approach combining enzyme activity and structure stability was adopted to evaluate the thermostability of GoxM4 and its mutants. After four rounds of computer-aided rational design, the best mutant, GoxM8, was obtained. The melting temperature (T m ) of GoxM8 was increased by 9 °C compared with GoxM4. The catalytic efficiency of GoxM8 was similar to GoxM4, suggesting that the enzyme activity-stability trade-off was counteracted. To explore its mechanism, we performed molecular dynamics simulations of GoxM4 and its mutants. Our findings provided a typical example for researching the enzyme activity-stability trade-off., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.

Key Message: The relative position of domains is critical for enzymatic properties of tau class glutathione S-transferases, and altering the position of linker far away from the active center affects catalytic property. Glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes whose main function is to improve plant resistance to stresses. To understand the structural effects of tau class GSTs on their function, using OsGSTU17 as an example, we predicted the residues involved in the interactions between its domains and linker region. We further detected the structural changes in mutants and the corresponding changes in terms of substrate activity and kinetic parameters. Four pairs of residues, including Ala14 and Trp165, Arg20 and Tyr154, Glu74 and Arg98, Asp77 and Met87, forming hydrogen bonds and salt bridges were found to play important roles in maintaining the relative position between the domains and linker region inside the protein. The hydrogen bond between Trp165 and Ala14 affected the structural stability has been demonstrated in our previous study. The mutant R20A lost almost all catalytic activity. Interestingly, the mutant E74A exhibited a significant decrease in activity towards 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole, 1-chloro-2, 4-dinitrobenzene and 4-nitrobenzyl chloride, while its activity towards substrate cumene hydroperoxide remained unchanged. Compared with other mutants, the mutant D77A exhibited decreased affinity to its substrates and increased activity towards 1-chloro-2, 4-dinitrobenzene and cumene hydroperoxide, but its thermodynamic stability did not change significantly. The relative position of individual domain was critical for enzymatic properties, and the linker which is far away from the active site could change the enzymatic properties of GSTs via altering the relative position of the individual domain. Our results provide insights into the relationship between structure and function of tau class GSTs.

Site-specific saturation mutagenesis within enzyme active sites can radically alter reaction specificity, though often with a trade-off in stability. Extending saturation mutagenesis with a range of noncanonical amino acids (ncAA) potentially increases the ability to improve activity and stability simultaneously. Previously, an Escherichia coli transketolase variant (S385Y/D469T/R520Q) was evolved to accept aromatic aldehydes not converted by wild-type. The aromatic residue Y385 was critical to the new acceptor substrate binding, and so was explored here beyond the natural aromatic residues, to probe side chain structure and electronics effects on enzyme function and stability. A series of five variants introduced decreasing aromatic ring electron density at position 385 in the order para-aminophenylalanine (pAMF), tyrosine (Y), phenylalanine (F), para-cyanophenylalanine (pCNF) and para-nitrophenylalanine (pNTF), and simultaneously modified the hydrogen-bonding potential of the aromatic substituent from accepting to donating. The fine-tuning of residue 385 yielded variants with a 43-fold increase in specific activity for 50 mm 3-HBA and 100% increased k cat (pCNF), 290% improvement in K m (pNTF), 240% improvement in k cat /K m (pAMF) and decreased substrate inhibition relative to Y. Structural modelling suggested switching of the ring-substituted functional group, from donating to accepting, stabilised a helix-turn (D259-H261) through an intersubunit H-bond with G262, to give a 7.8 °C increase in the thermal transition mid-point, T m , and improved packing of pAMF. This is one of the first examples in which both catalytic activity and stability are simultaneously improved via site-specific ncAA incorporation into an enzyme active site, and further demonstrates the benefits of expanding designer libraries to include ncAAs., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A large number of archaea live in hyperthermophilic environments. In consequence, their proteins need to adopt to these harsh conditions, including the enzymes that catalyze the synthesis of their membrane ether lipids. The enzyme that catalyzes the formation of the first ether bond in these lipids, geranylgeranylglyceryl phosphate synthase (GGGPS), exists as a hexamer in many hyperthermophilic archaea, and a recent study suggested that hexamerization serves for a fine-tuning of the flexibility - stability trade-off under hyperthermophilic conditions. We have recently reconstructed the sequences of ancestral group II GGGPS enzymes and now present a detailed biochemical characterization of nine of these predecessors, which allowed us to trace back the evolution of hexameric GGGPS and to draw conclusions about the properties of extant GGGPS branches that were not accessible to experiments up to now. Almost all ancestral GGGPS proteins formed hexamers, which demonstrates that hexamerization is even more widespread among the GGGPS family than previously assumed. Furthermore, all experimentally studied ancestral proteins showed high thermostability. Our results indicate that the hexameric oligomerization state and thermostability were present very early during the evolution of group II GGGPS, while the fine tuning of the flexibility - stability trade-off developed very late, independent of the emergence of hexamerization., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX-resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6-fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0-fold), 30 % (v/v) ethanol (2.1-fold), and 60 % (v/v) methanol (2.4-fold) compared with wild-type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β-hydrolase fold., (© 2020 Wiley-VCH GmbH.)

Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 β-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å 2 β-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

Halotolerant lipolytic enzymes have gained growing interest, due to potential applications under harsh conditions, such as hypersalinity and presence of organic solvents. In this study, a lipolytic gene, est56 , encoding 287 amino acids was identified by functional screening of a compost metagenome. Subsequently, the gene was heterologously expressed, and the recombinant protein (Est56) was purified and characterized. Est56 is a mesophilic (T opt 50 °C) and moderate alkaliphilic (pH opt 8) enzyme, showing high thermostability at 30 and 40 °C. Strikingly, Est56 is halotolerant as it exhibited high activity and stability in the presence of up to 4 M NaCl or KCl. Est56 also displayed enhanced stability against high temperatures (50 and 60 °C) and urea (2, 4, and 6 M) in the presence of NaCl. In addition, the recently reported halotolerant lipolytic enzymes were summarized. Phylogenetic analysis grouped these enzymes into 13 lipolytic protein families. The majority (45%) including Est56 belonged to family IV. To explore the haloadaptation of halotolerant enzymes, the amino acid composition between halotolerant and halophilic enzymes was statistically compared. The most distinctive feature of halophilic from non-halophilic enzymes are the higher content of acidic residues (Asp and Glu), and a lower content of lysine, aliphatic hydrophobic (Leu, Met and Ile) and polar (Asn) residues. The amino acid composition and 3-D structure analysis suggested that the high content of acidic residues (Asp and Glu, 12.2%) and low content of lysine residues (0.7%), as well as the excess of surface-exposed acidic residues might be responsible for the haloadaptation of Est56.

Hyaluronic acid (HA) is an anionic linear polysaccharide abundantly distributed in the extracellular matrix of mammalian connective, growing, and tumor tissues. Hyaluronidase is used as an important drug diffusion promoter and a tool enzyme to produce HA oligosaccharides. However, there is no thermostable hyaluronidase suitable for application to date. In this study, a thermophilic hyaluronate lyase, TcHly8C, from Thermasporomyces composti DSM22891 was expressed in Escherichia coli. The recombinant TcHly8C was most active at 70 °C, and it retained about 30% of initial activity after incubation at 60 °C for 28 days. The half-lives of TcHly8C at 60 °C and 70 °C were 16.1 d and 2.3 h, respectively. The optimum pH of TcHly8C is 5.93, and it was stable at pH 6.15-10.90. The presence of Mg 2+ could enhance its enzymatic activity significantly. K m , k cat , and k cat /K m of TcHly8C towards HA were 3.69 mg∙ml -1 , 17.82 s -1 , and 4.82 ml∙mg -1 ∙s -1 , respectively. TcHly8C degraded HA in an exolytic mode, and the end product was unsaturated HA disaccharide (ΔUA-GlcNAc). Overall, our results show that TcHly8C is the first reported PL8 exo-type hyaluronate lyase with high thermostability, which provides a potential enzyme used in medicine and production of HA oligosaccharides., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Endoglucanases provide an attractive avenue for the bioconversion of lignocellulosic materials into fermentable sugars to supply cellulosic feedstock for biofuels and other value-added chemicals. Thermostable endoglucanases with high catalytic activity are preferred in practical processes. To improve the thermostability and activity of the thermostable β-1,4-endoglucanase CTendo45 isolated from the thermophilic fungus Chaetomium thermophilum, structure-based rational design was performed by using site-directed mutagenesis. When inactivated mutation of the unique N-glycosylation sequon (N88-E89-T90) was implemented and the conserved Y173 residue was substituted with phenylalanine, a double mutant T90A/Y173F demonstrated enzymatic activity that dramatically increased 2.12- and 1.82-fold towards CMC-Na and β-D-glucan, respectively. Additionally, T90A/Y173F exhibited extraordinary heat endurance after 300 min of incubation at elevated temperatures. This study provides a valid approach to the improvement of enzyme redesign protocols and the properties of this endoglucanase mutant distinguish it as an excellent candidate enzyme for industrial biomass conversion., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Exo-inulinases are members of the glycoside hydrolase family 32 and function by hydrolyzing inulin into fructose with yields up to 90-95%. The N-terminal tail contributes to enzyme thermotolerance, which plays an important role in enzyme applications. However, the role of N-terminal amino acid residues in the thermal performance and structural properties of exo-inulinases remains to be elucidated. In this study, three and six residues of the N-terminus starting from Gln23 of the exo-inulinase InuAGN25 were deleted and expressed in Escherichia coli . After digestion with human rhinovirus 3 C protease to remove the N-terminal amino acid fusion sequence that may affect the thermolability of enzymes, wild-type RfsMInuAGN25 and its mutants RfsMutNGln23Δ3 and RfsMutNGln23Δ6 were produced. Compared with RfsMInuAGN25, thermostability of RfsMutNGln23Δ3 was enhanced while that of RfsMutNGln23Δ6 was slightly reduced. Compared with the N-terminal structures of RfsMInuAGN25 and RfsMutNGln23Δ6, RfsMutNGln23Δ3 had a higher content of (1) the helix structure, (2) salt bridges (three of which were organized in a network), (3) cation-π interactions (one of which anchored the N-terminal tail). These structural properties may account for the improved thermostability of RfsMutNGln23Δ3. The study provides a better understanding of the N-terminus-function relationships that are useful for rational design of thermostability of exo-inulinases.

A novel xylanase gene xynBCA, encoding a polypeptide of 439 residues (XynBCA), was cloned from Caldicoprobacter algeriensis genome and recombinantly expressed in Escherichia coli BL21(DE3). The amino acid sequence analysis showed that XynBCA belongs to the glycoside hydrolase family 10. The purified recombinant enzyme has a monomeric structure of 52 kDa. It is active and stable in a wide range of pH from 3 to 10 with a maximum activity at 6.5. Interestingly, XynBCA was highly thermoactive with an optimum temperature of 80 °C, thermostable with a half-life of 20 min at 80 °C. The specific activity was 117 U mg -1 , while the K m and V max were 1.247 mg ml -1 , and 114.7 μmol min -1  mg -1 , respectively. The investigation of XynBCA in kraft pulp biobleaching experiments showed effectiveness in releasing reducing sugars and chromophores, with best achievements at 100 U g -1 of pulp and 1 h of incubation. The comparative molecular modeling studies with the less thermostable Xylanase B from Clostridium stercorarium, revealed extra charged residues at the surface of XynBCA potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A that induces the accumulation of the substrate globotriaosylceramide. Currently approved enzyme replacement therapy using recombinant human α-galactosidase A improves patient symptoms but a majority of patients experience adverse events due to the multiple infusions required for full therapeutic efficacy. Our approach is to use medicinal chemistry and phylogenic comparisons to introduce mutations into the human enzyme to increase catalytic activity and/or stability to generate an improved therapeutic enzyme that may require fewer infusions. We designed mutations at three regions of the human α-galactosidase A: the active site, the dimer interface, and a site for glycosylation. The M208E mutation, adjacent to the Y207 active site residue, increased enzyme activity 3.01-fold. This mutation introduced a charged Glu residue that is adjacent to the Y207 active site residue and close to a site of N-glycosylation. The W277C mutation, designed to promote dimer stability, introduced a strong thiol-aromatic interaction (Cys-Phe) at the dimer interface and increased activity 2.31-fold. The W277C and M208E mutations modify the structure of the enzyme into forms with enhanced thermal stability 3.7- and 3.9-fold, respectively and positive cooperativity resulting in increased Hill coefficient from 1.0 to 4.60 and 3.47, respectively. Enhanced thermal stability and positive cooperativity predict improved in vivo activity and superior therapeutic properties. Our results demonstrate the value of in vitro mutagenesis for α-galactosidase A and support future perspectives to validate these results in Fabry disease patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d-galactose to d-tagatose at high temperature. l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His) 6 -tagged protein, immobilized on a copper-chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d-galactose to d-tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support., (© 2020 American Institute of Chemical Engineers.)

A novel GH1 β-glucosidase gene (bgla) from marine bacterium was sequenced and expressed in Escherichia coli. After purification by Ni 2+ affinity chromatography, the recombinant protein was characterized. The purified recombinant enzyme showed maximum activity at 40 °C, pH 7.5 and was stable between temperatures that range from 4 to 30 °C and over the pH range of 6-10. The enzyme displayed a high tolerance to glucose and maximum stimulation at the presence of 100 mM glucose. To improve glucose tolerance of the enzyme, a site-directed mutation (f171w) was introduced into β-glucosidase. The recombinant F171W showed a higher glucose tolerance than the wild type and maintained more than 40% residual activity at the presence of 4 M glucose. Additionally, the recombinant enzymes showed notable tolerance to ethanol. These properties suggest the enzymes may have potential applications for the fermentation of lignocellulosic sugars and the production of biofuels.

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 ( Pt NHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature ( T m ) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type Pt NHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

In a directed evolution aimed at improving enzymatic activity, a situation occurs where highly active variants can no longer be obtained from a template protein because the template is already located at a peak (local maximum) in the fitness landscape of activity for the sequence space. To overcome this situation, the template needs to descend the mountain (lose activity) once and climb another higher mountain. However, there is no solid guideline of how the template should go down. Here, we propose a stability index. Previous studies have shown that protein evolution is potentially governed by stability, and that proteins with low activity but high stability are more favorable templates for producing highly active variants. In our earlier works on conventional directed evolution by random mutagenesis of an esterase from Sulfolobus tokodaii, we identified variants with 3-fold higher activity than the wild-type as the highest activity variants. In this work, as a first step, stability-keeping variants were selected by five rounds of random mutagenesis and screening based on halo formation assay using the substrate tributyrin at 70 °C after heat treatment for 30 min at 90 °C. These variants are likely to be scattered at the feet of various mountains in the fitness landscape. Next, these variants were pooled and used as parental proteins for a conventional experiment with activity-based selection, where the activity of variants was assayed using their cell-free extracts on the substrate p-nitrophenyl butyrate at 75 °C. After two rounds of random mutagenesis, we successfully obtained a variant with 9-fold higher activity than the wild-type. These results indicate that the two-step selection by stability and activity enables us more easily to produce markedly activity-improving variants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A percentage of colorectal cancer (CRC) patients display low sensitivity to radiotherapy, which affects its therapeutic effect. Cancer cells DNA double-strand breaks (DSBs) repair capacity is crucial for radiosensitivity, but the roles of long noncoding RNAs (lncRNAs) in this process are largely uncharacterized. This study aims to explore whether lnc-RI regulates CRC cell growth and radiosensitivity by regulating the nonhomologous end-joining (NHEJ) repair pathway. CRC cells in which lnc-RI has been silenced showed lower cell growth and higher apoptosis rates due to increased DSBs and cell cycle arrest. We found that miR-4727-5p targets both lnc-RI and LIG4 mRNA and inhibit their expression. CRC cells showed increased radiosensitivity when lnc-RI was silenced. These results reveal novel roles for lnc-RI in both DNA damage repair and radiosensitivity regulation in CRC cells. Our study revealed that lnc-RI regulates LIG4 expression through lnc-RI/miR-4727-5p/LIG4 axis and regulates NHEJ repair efficiency to participate in DNA damage repair. The level of lnc-RI was negatively correlated with the radiosensitivity of CRC cells, indicates that lnc-RI may be a potential target for CRC therapy. We also present the first report of the function of miR-4727-5p.

Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (AfCel6A). Structure-based variants of AfCel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5-7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

β-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of β-glucanases in probiotics. Here, β-glucanase genes were cloned from Bacillus licheniformis, Lactobacillus fermentum and L. johnsonii. β-glucanase genes, as blg, lfg and ljg isolated from B. licheniformis, L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that β-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of α-helices and β-strands in the three mutants were altered, which might result in β-glucanase activity increasement. Analysis of β-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30-50 ℃ than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about β-glucanase-encoding genes in L. fermentum and L. johnsonii. These findings provide an efficient way to improve the activity of β-glucanase.

Thermostability is a fundamental characteristic of enzymes that is of high importance for industrial implementation of enzymatic catalysis. Cellobiohydrolases are enzymes capable to hydrolyze the most abundant natural polysaccharide - cellulose. These enzymes are widely applied in industry for processing of cellulose containing materials. However, structural and functional engineering of cellobiohydrolases for improving their properties is a challenging task. In this study, the thermostability of Penicillium verruculosum Cel7A cellobiohydrolase was increased through rational design of substitutions with proline. The stabilizing substitution G415P resulted in 3.4-fold increase in half-life time at 60 °C compared to wild-type enzyme. Molecular dynamics simulations indicated a clear effect of the stabilizing substitution G415P and the destabilizing substitutions D62P, S191P, and S273P on the stability of the enzyme tertiary structure. The stabilizing substitution G415P decreased flexibility of the lateral sides of the enzyme active site tunnel, while the considered destabilizing substitutions increased their flexibility., 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. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed at high levels in human alveolar type II (ATII) cells in the lung. A common polymorphism causing an amino acid substitution (K333Q) was previously linked to a loss of LCAD antigen in the lung tissue in sudden infant death syndrome. However, the effects of the polymorphism on LCAD function has not been tested. The present work evaluated recombinant LCAD K333Q. Compared to wild-type LCAD protein, LCAD K333Q exhibited significantly reduced enzymatic activity. Molecular modeling suggested that K333 is within interacting distance of the essential FAD cofactor, and the K333Q protein showed a propensity to lose FAD. Exogenous FAD only partially rescued the activity of LCAD K333Q. LCAD K333Q protein was less stable than wild-type when incubated at physiological temperatures, likely explaining the observation of dramatically reduced LCAD antigen in primary ATII cells isolated from individuals homozygous for K333Q. Despite the effect of K333Q on activity, stability, and antigen levels, the frequency of the polymorphism was not increased among infants and children with lung disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis ( T. vaginalis ). The glucose-6-phosphate dehydrogenase ( g6pd ) gene from T . vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tv g6pd :: 6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP + ) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP + were performed. These results revealed that the protein becomes more stable in the presence of the NADP + . In addition, we determined the dissociation constant for the binding ( K d ) of NADP + in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half-life of 10 min at 70°C. It is a Ca 2+ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α-amylases, AmyKS presents a high affinity toward soluble starch with a K m value of 0.252 mg ml -1 . Coupled with the size-exclusion chromatography data, MALDI-TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non-maltogenic α-amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme., (© 2020 American Institute of Chemical Engineers.)

In the present investigation, a lipid hydrolyzing gene RPK01 was cloned from metagenome source of hot spring. Expression and purification of recombinant protein revealed single purified protein band of ~24 KDa on 12% SDS-PAGE, and is well corroborated with the deduced molecular weight of protein as calculated from its amino acid sequence. The purified protein displayed high activity towards short chain fatty acids and was found to be completely stable at 30°C till 3h, it further retained ~40% activity at 50°C and 60°C temperature till 3h. Additionally, the pH stability assay showed its functionality in broad range of pH, with maximum stability observed at pH 2.0, it decreases from pH 4.0 to pH 12.0, and nearly showed 40% activity in these pH values. Both circular dichroism and intrinsic Trp fluorescence studies revealed conformational stability of protein structure at wide range of temperature and pH. Enzyme activity enhances in presence of non-ionic surfactants like Tween 20 and TritonX-100. Further, inhibitors of the active site residues including PMSF and DEPC alone were unable to inhibit enzyme activity, while cumulative presence of calcium and inhibitors reduces enzyme activity to 90%, indicating conformational changes in the protein. Molecular simulation dynamics analysis revealed a calcium binding site near the lid helix of this protein(Asn75-Ile80)., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cytochrome bd-I oxidase is a terminal reductase of bacterial respiratory chains produced under low oxygen concentrations, oxidative stress, and during pathogenicity. While the bulk of the protein forms transmembrane helices, a periplasmic domain, the Q-loop, is expected to be involved in binding and oxidation of (ubi)quinol. According to the length of the Q-loop, bd oxidases are classified into the S (short)- and the L (long)-subfamilies. Here, we show that either shortening the Q-loop of the Escherichia coli oxidase from the L-subfamily or replacing it by one from the S-subfamily leads to the production of labile and inactive variants, indicating a role for the extended Q-loop in the stability of the enzyme., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Trehalose synthase converts maltose into trehalose in a single conversion step via intramolecular transformation and is thus useful for industrial production. In this study, we synthesized a thermophilic trehalose synthase from Thermus antranikianii (TaTS), which was recombinantly expressed in Escherichia coli BL21(DE3). The recombinant TaTS showed the highest activity at pH 7.0 and 60°C, with the maximum trehalose yield (76.8%) obtained at pH 7.0 and 30°C. TaTS activity was stable over a wide pH and temperature range of 6-10 and 4-70°C, respectively, over 6 h of incubation. The enzyme activity was strongly inhibited by Co 2+ , Cu 2+ , Zn 2+ , sodium dodecyl sulfate, and Tris. TaTS showed a 1.48-fold higher catalytic efficiency (k cat /K m ) for maltose than for trehalose. Overall, these results demonstrate the good application potential of the recombinant enzyme TaTS in the efficient conversion of trehalose from maltose, with superior environmental tolerance to other trehalose synthases reported., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.