Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M−1 s−1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from  Brevundimonas diminuta MG (bdPTE) [bdPTE has exhibited hydrolytic activities against a wide range of OP insecticides such as ethyl-paraoxon, parathion, methyl parathion, dursban, coumaphos, diazinon, fensulfothion, and cyanophos. The catalytic efficiency (kcat/KM) towards paraoxon is particularly high, reaching at 4.0 × 107 M−1s−1 [bdPTE demonstrated the ability to degrade both G-series  and V-series (VX and VR) nerve gases [bdPTE have shown a potential of these enzymes to apply for practical decontamination of toxic OP compounds. On the other hand, it is inevitable that OP-degrading enzymes must possess the stability under wide range of temperatures, pH, and the presence of organic solvents, in consideration of the real decontamination conditions of OP-contaminated soil, surfaces, air, water and waste.The ever-increasing number of organophosphate (OP) pesticides poisoning, as well as intensive use of OP chemical warfare agents (CWA) in civil war and terrorist acts have urged the immediate establishment of sustainable and reliable methods for sensing, decontamination and detoxification of OP compounds ,2. Enzym (bdPTE) . bdPTE h7 M−1s−1 ,14,15,16ve gases ,14,15,16bdPTEs, being classified in the same amidohydrolase superfamily. Several thermostable PLL enzymes have been identified from either hyperthermophilic archaea or thermophilic/extremophilic bacteria [In this context, our group has focused on the studies of an enzyme family of lactonases with promiscuous phosphotriesterase activity, dubbed Phosphotriesterase-Like-Lactonases (PLLs) ,20,21,22bacteria ,25,26,27Deinococcus radiodurans was improved toward two OP pesticides, ethyl and methyl paraoxon, through both structure-based and random mutagenesis approaches [Geobacillus kaustophilus by stepwise loop insertion strategy (StLois), in which randomized residue pairs were inserted in loop 7 of GKL in a stepwise manner. Obtained new variant, ML7-B6, has shown a catalytic efficiency of 2.4 × 105 M−1 s−1 at 65 °C against paraoxon [A variety of both rational and directed evolution approaches have been applied on thermostable PLL enzymes and yielded engineered PLL variants. For example, hydrolytic activity of DrOPH from proaches . More reparaoxon .SsoPox variants generated by directed evolution and semi-rational design [SsoPox triple mutant, C258L/I261F/W263A (3Mut), has shown catalytic efficiency (kcat/KM) for paraoxonase activity approaching 4.5 ± 0.8 × 104 M−1 s−1 at 65 °C. We speculated that the improvement in 3Mut is presumably due to the combination of the enlargement of active site by the alanine substitution at position 263, accompanied by the hydrophobic patch created by both leucine and phenylalanine at positions 258 and 261, respectively. These three positions are among the 16 amino acid residues identified through a structural alignment between SsoPox and bdPTE, which revealed the involvement of these 16 residues in the lactone substrate binding site. Furthermore, we performed site-saturated mutagenesis by means of smart library construction on 6 additional residues  that were identified through a bioinformatics approach. However, we did not obtain variants with better paraoxonase activity than 3Mut [SsoPox 3Mut (αsA1) and other variants showing enhanced phosphotriesterase activity [SsoPox, confirming our hypothesis obtained from the model analysis [Previously our group reported the characterization of l design ,22. In al design ,29. Receactivity . It was analysis .SsoPox. The obtained new mutant, V82L/C258L/I261F/W263A (4Mut), exhibited catalytic efficiency at 1.6 × 105 M−1 s−1 against paraoxon hydrolysis at 70 °C.In the present study, starting from 3Mut, we have designed a new directed evolutionary approach based on DNA StEP gene recombination method for further improvement of promiscuous phosphotriesterase activity of We further performed in-depth structural analyses by solving 3D structure, computational as well as circular dichroism (CD) analyses in search for the structural determinants for the improved catalytic activity in 4Mut. Here we demonstrate that a single mutation not located at the active site can alter the dimer flexibility through reorganization of dimer conformation. The increased dimer flexibility could further bring about the improvement in hydrolytic activity. This hypothesis can be also applicable for molecular evolution of this class of enzymes.DNA StEP recombination method generally requires more than one kind of homologous genes as templates for the syntheses of polynucleotides . During SsoPox triple mutant C258L/I261F/W263A (3Mut), sislac from Sulfolobus islandicus, sacpox from Sulfolobus acidocaldarius, vmolac from Vulcanisaeta moutnovskia, gkl from Geobacillus kaustophilus, and dr0930 from Deinococcus radiodurans as template genes for recombination. Sequence alignments of DNA and encoded proteins from these 6 genes are shown in At the first stage of the generation of PLL mutant library, we utilized 6 PLL genes, including E. coli BL21 (DE3) expressing mutant PLL enzymes was established as shown in Prior to screening for variants with improved paraoxonase activity, esterase activity assay was carried out as primary screening. In situ esterase activity assay for More than 20,000 colonies were analyzed by primary esterase activity assay. Among them, colonies showing high esterase activity were selected for secondary screening for phosphotriesterase activity assay in 96-well plates using paraoxon as substrate. Paraoxonase activity of these variants was compared with that in 3Mut. Approximately 2000 variants were screened by paraoxonase activity assay, however, we were not able to identify variants showing higher paraoxonase activity than that of 3Mut.For the verification of gene recombination in this study, we selected four mutants  for further investigation. The comparison of paraoxonase activity between these 4 variants and 3Mut is shown in dr0930 and gkl, as templates to recombine with SsoPox triple mutant C258L/I261F/W263A for the generation of new variants. These two bacterial PLL genes show low sequence identities with respect to C258L/I261F/W263A. GKL shows 46.7% identities at DNA level, and 35.8% at protein level. Dr0930 exhibits even lower sequence identities against 3Mut, which were 42.6% and 29.9% at DNA and protein level, respectively  under the presence of different metal ions including Co2+, Mn2+, Zn2+, Ni2+ or Cd2+. The effect of different metals in the growth medium on the expression level of 4Mut was analyzed by SDS-PAGE. By observing the signals at around 35 KDa, which corresponds to the molecular mass of 4Mut monomer, it was confirmed that there was no significant difference in the expression level of 4Mut in the absence or presence of different metal ions  at a medium scale (1.5 L) and purified under the presence of 0.5 mM CoCl2 in the buffer solution throughout the purification process. In order to exclude any influence on enzymatic characterization caused by contaminating proteins, we only choose the fraction containing 4Mut with more than 95% purity after size-exclusion chromatography as the final step of the purification. 0.6 mg of highly pure (> 95%) 4Mut was obtained from 1.5 L cell culture and utilized for the enzymatic characterization.For further characterization, 4Mut was expressed in 2HPO4/NaH2PO4, HEPES-NaOH and Gly-NaOH) were utilized. The absorbance was measured at 348 nm, the pH-independent isosbestic point between p-nitrophenol and p-nitrophenolate ion. As shown in SsoPox and its single mutant [The pH dependency of 4Mut on paraoxonase activity was examined at 70 °C. In order to cover a wide range of pH from 5.0 to 9.5, three different kinds of buffers  at each temperature showed that 4Mut retains half of its paraoxonase activity for 16.7 h at 65 °C, and 8.4 h at 70 °C  [We assessed the thermal stability of 4Mut by measuring paraoxonase activities after incubating the enzyme for certain amount of time up to 4 h at different temperatures . at 70 °C d. At 75 ctively) .SsoPox wild-type [SsoPox and its mutants. The specific activity of 4Mut against paraoxon reached 206.4 ± 18.3 U/mg at 70 °C. This value is about 500-fold improved compared with SsoPox [kcat and KM values, 4Mut showed 2-fold improvement in both parameters with respect to 3Mut. As a result, catalytic efficiency (kcat/KM) reached 1.6 × 105 M−1 s−1, which is more than 3.5-fold and 42-fold improved in comparison with 3Mut and SsoPox, respectively.Even though PLL enzymes exhibit significant phosphotriesterase activities particularly on paraoxon, they are natural lactonases that catalyze the hydrolysis of lactones and acyl-homoserine lactones (AHLs) as their main function . In addiild-type . In parth SsoPox ,22. In tkcat and KM values of 4Mut for the hydrolysis of methyl-paraoxon were determined as 30.1 s−1 and 0.871 mM, respectively, resulting in kcat/KM with 3.5 × 104 M−1 s−1.On the other hand, hydrolytic activity of 4Mut on methyl-paraoxon (50.9 ± 12.0 U/mg) was slightly decreased in comparison with that of 3Mut obtained at pH 8.5, 65 °C. The kcat, KM and kcat/KM for the paraoxonase activity of 4Mut at 25 °C were 33.0 s−1, 0.645 mM and 5.1 × 104 M−1 s−1, respectively. The catalytic efficiency was improved about 2.8-fold compared with the value obtained for 3Mut at 25 °C [Kinetic parameters for hydrolytic activity of 4Mut against paraoxon were also examined at 25 °C . The detat 25 °C .SsoPox and its mutants have been strongly enhanced by anionic detergents such as SDS and DOC [SsoPox and 3Mut increased 17-fold (at 70 °C) and 2-fold (at 65 °C) under the presence 0.025% of SDS, respectively [kcat/KM) at 70 °C reached 4.3 × 105 M−1 s−1, exhibiting 2.6-fold enhancement under the presence of 0.025% SDS. Both lowered KM and increased kcat by SDS were found to be responsible for the improved kcat/KM value. At 25 °C, while kcat value was improved slightly, KM value was reduced approximately 2-fold under the presence of 0.025% SDS, resulting in 2-fold improvement in kcat/KM (1.0 × 105 M−1 s−1).Previously it was reported that promiscuous phosphotriesterase activity of  and DOC ,22,32. TWe further investigated the stability of 4Mut by incubation in the presence of 0.025% SDS at room temperature for certain period of times b. 4Mut rIn contrast to the improved hydrolytic activities on phosphotriesters, 4Mut shows no catalytic activities on carboxylesters including pNP-butanoate and β-naphthyl acetate. High catalytic activity on synthetic lactone TBBL (5-thiobutilγbutyrolactone) was detected (63.0 U/mg), however, there was no activity on hydrolysis of C8-HSL and C10-HSL (N-octanoyl and N-decanoyl homoserine lactones). Overall, 4Mut possesses similar substrate profile with 3Mut .SsoPox, W263F and SacPox to hydrolyze a deadly nerve agent, cyclosarin [We previously demonstrated the ability of closarin . In addiclosarin . It is oE. coli culture for the enzyme preparation. This is in contrast with the previous result in which SsoPox was shown to possess heterobinuclear metal center constituted by an iron at α-site and a cobalt cation at β-site after purification in the presence of Co2+ ion in the buffer [2+; red spheres) detected. The RMS difference between 3Mut and 4Mut was 0.59 Å on 604 superimposed Calpha atoms. In 4Mut, many small changes with respect 4Mut were visible. In particular an hydrogen bond between Tyr99 and His 170 coordinating one of Co2+ ions was missing. Many residues were shifted with respect the 3Mut. That suggests a different conformation imparted by mutations.3D structure of 4Mut was obtained by the hanging drop method as described in Methods. At first we got crystals from 10% PEG 3000 0.05 M HEPES pH 7.5. These crystals did not diffract at high resolution. Thus we used seeds of these crystals and got crystals using the following conditions: 10% PEG 3000, 0.05 M HEPES pH 7.5, 0.1 M NaCl. These crystals were obtained by utilizing the sitting drop vapor diffusion method, at 19°C and diffracted at 2.8 Å . Detailse buffer . Howevere buffer . The detSsoPox, 3Mut and 4Mut (SsoPox (PDB code: 2VC7) exhibited two large grooves at the interface of two monomers. Importantly, amino acid residues lined along these cavities include Val 27, Leu 72, Tyr 97, Ile 98, and Trp 263, which were all previously reported in the mutagenesis study to increase the phosphotriesterase activity of the enzyme because of their involvement in the catalysis based on the comparison with PTE [SsoPox structure appeared as a large groove alongside the interface of two monomers . Since central groove is located at the interface of two monomers in an enzyme molecule, the larger space between two monomers can be interpreted as higher flexibility of the dimer. From this view, we could conclude that SsoPox has the lowest dimer flexibility among the three enzymes, with more compact, rigid conformation. Both mutations introduced in 3Mut and 4Mut showed positive effects on the central groove volume, therefore on dimer flexibility.Surprisingly, the single mutation introduced in 4Mut confirmed by the structure is not located in the vicinity of active site a. In ordand 4Mut . The dimwith PTE ,22,34. Twith PTE . In the monomers c,d. We pand 4Mut a. The ceIt is of great interest to investigate how a single mutation located far from the active site affected the paraoxonase activity. Our hypothesis is that, single mutation at position 82 has slightly altered the overall architecture of the enzyme, which resulted in the improved catalytic activity. We further investigated the structural organization of 3Mut and 4Mut by far- and near-UV circular dichroism analyses.SsoPox, suggesting that two mutant enzymes possess a large alpha-helical content as wild-type enzyme does, and that mutations did not collapse the secondary structure of the enzyme [The far-UV CD spectra of 3Mut and 4Mut are shown in e enzyme . At 75 °. Thermal denaturation curves revealed that denaturation temperatures for 4Mut and 3Mut were 86.7 and 92.3 °C, respectively. 3Mut exhibited higher melting temperature than 4Mut by 5.6 °C, however, these values are much lower than that of SsoPox, which was previously determined as 106 °C [For further investigation on the conformational stability of two enzymes at the secondary structure level, we acquired thermal denaturation curves by recording the molar ellipticity at 222 nm c. Thermas 106 °C .CD spectra at near-UV region were also acquired for both 3Mut and 4Mut at different temperature conditions . It is nSsoPox 3Mut (C258L/I261F/W263A), GKL and Dr0930 to generate new mutant PLL variants through DNA StEP method. Two bacterial PLL genes, gkl and dr0930, show quite low sequence identities with respect to C258L/I261F/W263A; 35.8% for gkl and 29.9% for dr0930, respectively. In principle, DNA StEP method requires homologous genes with more than 80% sequence identities for recombination [We utilized three homologous PLL genes encoding bination . TherefoSsoPox to 3Mut, we observed a weak trade-off in the specificity between main lactonase activity and promiscuous paraoxonase activity. Three mutations brought about a 9-fold decrease in lactonase activity on TBBL, while paraoxonase activity increased 292-fold. This led to the paraoxonase activity in 3Mut exhibiting approximately 3-fold higher than the lactonase activity on TBBL, in contrast to the wt, which showed 760-fold lower paraoxonase activity than its main lactonase activity [In the course of molecular evolution from activity . In the activity . It is lactivity .SsoPox variants suggested that conformational rearrangement in active site loop 8, which is involved in substrate binding, is strongly correlated with substrate specificity spectrum [As shown in spectrum . Destabikcat/KM values for the paraoxonase activity of both 4Mut and 3Mut increased at higher temperature with respect to those at 25 °C, different factors are involved in their enhancement. In case of 4Mut, the substrate affinity (KM) did not show significant changes between 25 and 70 °C. The higher kcat/KM value at 70 °C was therefore mainly due to the increased kcat. In contrast, KM in 3Mut at 25 °C showed a significantly reduced value compared with that obtained at 65 °C. However, specific activity (Vmax) of 3Mut at 25 °C was considerably low (8.74 U/mg) compared with the value at 65 °C (123.6 U/mg), leading to the low kcat for 3Mut at 25 °C (6.91 s−1). It seems that positive effect of KM value on catalytic efficiency in 3Mut was masked by low kcat at 25 °C. In addition, it should be noted that 4Mut showed approximately 3-fold increased kcat/KM values in comparison with 3Mut at their optimal temperature (70 °C or 65 °C) and 25 °C. At high temperatures, both improved substrate affinity and kcat have contributed to the enhanced catalytic efficiency in 4Mut. By contrast, at 25 °C, even though 3Mut exhibited better KM compared with 4Mut, kcat was almost 5-fold lower than the value in 4Mut. Therefore, in comparison with 3Mut, high kcat in 4Mut seems the primary factor in enhanced kcat/KM at 25 °C. Furthermore, kinetic data obtained in the presence of SDS revealed that 4Mut responded in a different way to the 0.025% SDS at 70 °C with respect to 3Mut. Even though 0.025% SDS enhanced the catalytic efficiency of 3Mut by 2-fold at 65 °C, improved kcat/KM was mainly due to a significant improvement of the substrate affinity that compensated a large reduction in the kcat value.On the other hand, we observed that single mutation introduced in 4Mut has resulted in altered enzymatic and biochemical properties compared with 3Mut. First of all, kinetic comparisons of paraoxonase activities between 4Mut and 3Mut at two temperatures (25 °C and either 65 or 70 °C) pointed out some interesting differences. In fact, even though From the biochemical point of view, 4Mut exhibited a characteristic pattern of thermophilicity. 4Mut did not show a clear optimal temperature, but rather maintained high activity in the temperature range between 55 °C and 70 °C. On the other hand, 3Mut showed a clear optimal temperature at 65 °C, with a substantial activation effect by increased temperature up to 65 °C . This heIn case of 4Mut, the rearrangement of tertiary structure can be due more probably to tryptophan and tyrosine moieties, which influence the CD signals at about 284 nm.Two residues have a different accessibility in this case, namely Trp36 and Tyr40. From far- and near-UV CD analyses, we could conclude that molecular evolution from 3Mut to 4Mut involving an improvement in paraoxonase activity is accomplished by structural rearrangement at tertiary structure level, which thus affects the quaternary organization, without significantly affecting the secondary organization of the enzyme.1/2 values were determined as 3.3 h and 2.1 h, respectively. Previously characterized single mutant of SsoPox, W263F, showed much lower thermal stability, with t1/2 values as 3.3h at 70 °C and 1.5h at 80 °C [SsoPox (106 °C), they are substantially higher than that for PTE from Brevundimonas diminuta MG (75 °C) [From the thermal stability analyses, it was revealed that 4Mut retains half of its paraoxonase activity for 16.7 h at 65 °C, and 8.4 h at 70 °C. At 75 and 80 °C, the tat 80 °C . On the  (75 °C) .SsoPox exhibited the lowest value among SsoPox, 3Mut and 4Mut . Molecular mass markers for SDS-PAGE, restriction enzymes were obtained from Bio Rad  and New England BioLabs , respectively. All other chemicals were purchased from Sigma Chemical Co. . Chemical nerve agents including tabun (GA), sarin (GB), soman (GD), cyclosarin (GF) and VX were provided by the Bundeswehr Institute of Pharmacology and Toxicology .E. coli strains TOP10 or BL21(DE3) were grown in Luria-Bertani (LB) medium containing either ampicillin (100 mg/mL) or kanamycin (50 mg/mL) at 37 °C. Purification of plasmid DNA was performed using QIAprep Miniprep or Midiprep kits . The concentration of DNA was determined by NanoDrop 1000 .Standard molecular cloning techniques were employed. sislac gene was performed as described below. A 945-bp of sislac gene fragment was amplified by PCR reaction using genomic DNA of Salfolobus islandicus M 16.4  as template. 300 ng of genomic DNA of S. islandicus M 16.4 was mixed with the PCR reaction solution containing 2 μM 5′ sislac oligonucleotide (5′-CTATTATCATATGAGAATACCATTAGTTGGG-3′), 2 μM 3′ sislac oligonucleotide (5′-ATATAAGCTTTTAGCTGAAGAATTTTTTCGG-3′), 200 μM dNTP, 2.0 mM MgSO4, 1U Vent DNA polymerase in the buffer conditions suggested by the manufacturer. The primer 5′ sislac was designed to introduce an NdeI restriction site upstream of the sislac gene, whereas primer 3′ sislac was designed to introduce a HindIII restriction site downstream from the stop codon of sislac gene. The amplification of sislac gene was performed by the PCR programme as follows: 2 min at 95 °C, followed by 30 cycles of 95 °C for 2 min, 58 °C for 1 min and 72 °C for 1 min. The amplified sislac gene was digested by NdeI and HindIII restriction enzymes, and purified by QIAquick PCR purification kit . Obtained DNA fragments were ligated into the vector pT7-7 (a derivative of pBR322). Vector pT7-7 was treated with Alkaline Phosphatase, Calf Intestinal (CIP), prior to ligation reactions. Chemical competent E. coli TOP10 (Stratagene) was transformed with ligated plasmid DNA and subsequently plated on LB agar plate containing 100 μg/mL ampicillin. Obtained plasmid DNA pT7-7-sislac was sequenced to verify the insertion of sislac gene.Cloning of vmolac was optimized for the expression in E. coli and cloned into vector pEX-A2 by Eurofins Genomics, Germany. NdeI and HindIII restriction sites were introduced upstream and downstream of vmolac gene, respectively. Chemical transformation of pEX-A2-vmolac into E. coli (TOP10) was carried out and plasmid DNA pEX-A2-vmolac was obtained through Midiprep kits .The gene encoding C258L/I261F/W263A and pT7-7-dr0930 constructs were obtained as described in Del Giudice et al. 2016 [gkap(gkl) construct was kindly donated by Yan Feng from Shanghai Jiao Tong University, China.The plasmid DNA pT7-7-sacpox was generated as described in Porzio et al. 2007 [al. 2016  and Mandal. 2016 , respectC258L/I261F/W263A construct was digested by NdeI and EcoRI restriction enzymes, while NdeI and BamHI were utilized to digest pT7-7-dr0930. For the digestion of pT7-7-dr0930, BamHI and HindIII were used. pT7-7-sislac and pEX-A2-vmolac were digested with NdeI and HindIII. pT7-7-sacPox was digested with NdeI and PstI. Digestion for vector-gene construct was performed at 37 °C for 1.5 h. Digested gene fragments (approximately 1000 bp) were gel-purified by QIAquick Gel Purification kit . Purified gene fragments were eluted with 40 µl sterile milliQ water and stored at −20 °C.pT7-7-4, 1U Vent DNA polymerase in appropriate buffer condition, 1 µM 5′ oligonucleotide (5′-CTATTATCATATGAGAATACCATTAG TTGGG-3′) and 1 µM 3′ oligonucleotide (5′-ATATAAGCTTTTAGCTGAAGAATTT TTTCGG-3′). The 5′ primer was designed to introduce an NdeI restriction site upstream of the C258L/I261F/W263A gene, whereas 3′ primer was designed to introduce a HindIII restriction site downstream from the stop codon of C258L/I261F/W263A gene. Total volume was adjusted at 50 µl by adding sterile milliQ water. Thermal cycle of DNA StEP programme included denaturation at 94 °C for 30 s, followed by extremely abbreviated annealing/polymerase-catalyzed extension at 55 °C 2 s. In order to obtain full-length mutant gene, the thermal cycle was repeated 50 times.1.0 ng of digested and purified PLL genes were mixed in the reaction solution containing 200 µM dNTP mix, 2.0 mM MgSO4, 1U Vent DNA polymerase in the buffer conditions suggested by the manufacturer. The amplification was performed by the PCR programme as follows: 2 min at 95 °C, followed by 30 cycles of 95 °C for 2 min, 58 °C for 1 min and 72 °C for 1 min.Recombinant product obtained through DNA StEP reaction was digested by DpnI restriction enzyme in order to eliminate template genes, and purified by QIAquick PCR purification kit . 100 ng of purified DNA fragments were amplified by PCR in a reaction solution containing 2 µM 5′ oligonucleotide (5′-CTATTATCATAT GAGAATACCATTAGTTGGG-3′), 2 µM 3′ oligonucleotide (5′-ATATAA GCTTTTAGCTGAAGAATTTTTTCGG-3′), 200 µM dNTP, 2.0 mM MgSOE. coli BL21 (DE3) by electroporation using the MicroPulser electroporator (BIO-RAD) with the voltage of 2.5 kV. Transformed E.coli BL21 (DE3) was subsequently plated on LB agar plate containing 100 µg/mL ampicillin.Amplified mutant library were then digested with NdeI and HindIII restriction enzymes. After purification using QIAquick PCR purification kit, mutant library were ligated into the vector pT7-7 which was treated with Alkaline Phosphatase, Calf Intestinal (CIP) prior to ligation reactions. 10 ng of ligations were used to transform 40 µL of electrocompetent p-nitrophenol, one of the hydrolysis products of paraoxon.Each colony on LB agar plates was picked up to inoculate 200 µL of LB medium containing 100 µg/mL ampicillin in the 96-well cell culture sterile plates. The plates were incubated for overnight at 37 °C and mutant PLL enzyme expression was induced for 3 h at 37 °C after addition of IPTG with the final concentration of 1 mM. For the cell lysis, 80 µL of cell culture was mixed with 20 µL of lysis buffer containing 0.1 M Tris-HCl (pH8.0) and 0.25% SDS in a new 96-well cell culture sterile plate, and incubated at 4 °C for 1 h. Identical two 96-well plates were prepared by transferring 50 µL of lysate solution into a new 96-well plate for the duplication of paraoxonase activity assay. 150 µL of activity assay solution was added to lysis solution with the final concentration of 20 mM Tris-HCl (pH8.0 at 70 °C) and 1 mM paraoxon as substrate. The absorbance at 405 nm was measured after incubation for 30 min at 70 °C, in order to detect the production of 2, (0.5 mM) were added to the growth medium and incubated at 37 °C for 4 h. Cells were harvested by centrifugation at 6000 rpm for 15 min at 4 °C, and pellets (4.5 g) were stored at −20°C. Wet frozen cells were thawed and resuspended in 25 mL of buffer A  and lysed by sonication (Branson). The programme for sonication was set as follow; 10 s ON, 10 s OFF, total time 3 min, 30% intensity. Cell debris was removed by centrifugation at 80,000 g for 30 min at 4 °C. Crude extract (25 mL) was incubated at 60 °C and 70 °C for 15 min under gentle stirring, in order to thermoprecipitate proteins derived from E. coli and separated by centrifugation at 80,000 g for 30 min at 4 °C between each thermoprecipitation step. Subsequently anion exchange chromatography was performed for further purification. Enzyme solution obtained through 70 °C incubation was loaded onto Q-Sepharose Fast Flow FPLC column (Pharmacia) equilibrated with buffer A, connected to an AKTA pure system . Following the wash with buffer A, a linear gradient of NaCl (0–0.5 M) was applied for the elution of the proteins with the flow rate 1 mL/min. The fractions were analyzed by both SDS-PAGE and phosphotriesterase activity assay using 1 mM paraoxon as substrate as described below. Fractions with activity on paraoxon were concentrated by ultrafiltration. Size-exclusion chromatography was performed using Superdex 75 column 16 × 60 cm2  equilibrated with buffer containing 20 mM HEPES-NaOH (pH 8.5), 0.5 mM CoCl2 and 200 mM NaCl. The flow rate was set as 0.5 mL/min. The fractions were analyzed by SDS-PAGE and paraoxonase activity assay as described below, and stored at 4 °C.3 mL of LB medium with 100 µg/mL ampicillin was inoculated with single colony, and incubated at 37 °C for 10 h. The 3 mL pre-culture was used to inoculate 1.5 L of LB medium containing 100 µg/mL ampicillin and cells were grown at 37 °C for overnight. IPTG (0.5 mM) and CoClp-nitrophenol in 1 cm path-length cells using Cary 100 spectrophotometer . The molar absorption coefficients used for p-nitrophenol were reported previously [Standard paraoxonase activity assay was performed essentially as described in Merone et al. 2005 . In brieeviously . Activiteviously  using 1 eviously . KineticE.coli BL21(DE3) carrying plasmid pT7-7-4Mut were grown in 100 mL LB medium containing 100 µg/mL ampicillin at 37 °C for overnight. 10 mL aliquots of cell culture were prepared in new sterile tubes. IPTG with the final concentration of 0.5 mM and either ZnCl2, CoCl2, MnCl2, CdCl2 or NiSO4  were added prior to further incubation at 37 °C for 4 h. Cells were harvested by centrifugation at 5000 rpm for 15 min at 4 °C, and pellets were stored at −20 °C. Each wet frozen cell was thawed, resuspended in 500 µl of 20 mM HEPES-NaOH (pH 8.5) and lysed by sonication (Branson). The programme was set as follow; 10 s ON, 10 s OFF, total time 1 min, 20% intensity. Cell debris was removed by centrifugation at 13,000 rpm for 10 min at 4 °C. Crude extract was incubated at 70 °C for 10 min and precipitated proteins derived from E. coli were removed by centrifugation at 13,000 rpm for 10 min at 4 °C. The paraosonase activity assay was performed as described above using each enzyme solution prepared in the presence of different metal ions.Effect of metal ions on paraoxonase activity of 4Mut was analyzed essentially described in Porzio et al. 2013 . In detaThe dependencies of paraoxonase activity on pH, temperature and thermal stability were measured as described in Merone et al. 2005 . The temLactonase activities on N-octanoyl homoserine lactone (C8-HSL), N-decanoyl homoserine lactones (C10-HSL) and 5-thiobutylγbutyrolactone (TBBL) were measured as described in Porzio et al. 2013 .p-nitrophenyl (pNP) butanoate and β-naphtyl acetate was performed as described in Merone et al. 2005 [Activity assay on carboxylesters including al. 2005 .E. coli BL21 (DE3) transformed with pT7-7-4Mut was grown in 7 L of LB medium containing 100 µg/mL ampicillin at 37 °C overnight. Protein expression was induced by 0.5 mM IPTG. Cell harvesting and purification were performed as described in the section “Protein expression and purification in large scale”. In addition, in order to obtain pure enzyme with high yield, additional purification process was performed after size-exclusion chromatography; the fractions containing 4Mut, with approximately more than 90% purity, were selected and pooled. Thermal precipitation was performed at 70 °C for 30 min, followed by ultracentrifugation at 80,000× g for 1 h at 4 °C. Supernatant was diluted 10-times with buffer A in order to lower the concentration of sodium chloride in the protein solution and applied on MonoQ column  equilibrated with buffer A, connected to an AKTA pure system. After washing with buffer A, proteins were eluted by a linear gradient of NaCl (0–0.5 M) with the flow rate 1 mL/min. Purity of obtained fractions was analyzed by SDS-PAGE and fractions with more than 95% purity were pooled. Pure enzyme solution was substituted with buffer solution containing 20 mM HEPES-NaOH (pH 8.5), 0.5 mM CoCl2 and 200 mM NaCl and concentrated through ultrafiltration. Pure 4Mut with final concentration of 6.0 mg/mL was used for crystallization.SsoPox-4Mut plated like monoclinic crystals were obtained using the sitting drop vapor diffusion method with a Mosquito robot (TTP LabTech) at 19 °C utilizing the precipitant 20% propanol (v/v), 20% Polyethylene glycol (PEG) 3000, 100 mM HEPES pH 7.5 and 0.2 M NaCl. The 4Mut crystals formed in the orthorhombic space group 12121P2, with two dimers per asymmetric unit. Data collection was performed under cryo conditions (100 K), on ID30A-3 at the European Synchrotron Radiation Facility . All diffraction images were indexed and integrated using the Mosflm program [SsoPox-4Mut was solved by molecular replacement with the program PHASER [SsoPox from the Saccharolobus solfataricus (PDB-ID: 4KES); [obs-DFcalc, and mFobs-DFcalc maps using COOT [free value. All models were optimized using PDB_REDO [ program , and the program . Structu program  from them PHASER , using t: 4KES); . All ste: 4KES); . The moding COOT . RefinemPDB_REDO , and werPDB_REDO . DetailsMetal content analysis was performed by ICP-MS as reported in Porzio et al. 2013 .1 which was then plotted versus time to get the detoxification constant kobs. Data were corrected for spontaneous OP hydrolysis and finally kcat/KM was calculated. Assays were performed in duplicate, and results were the means of two independent experiments.5 µL of 1.0 mg/mL 4Mut in 20 mM HEPES pH 8.0 200 mM NaCl was mixed with 300 µL of nerve agent  in 0.1 M Tris-HCl pH 7.4 at 37 °C. 50 µL aliquot was added in cuvette containing 3000 µL of 0.1 M phosphate buffer pH7.4, 100 µL of 10 mM DTNB and 50 µL of 28.4 mM acetylthiocholine at different times (5 to 60 min). The reaction was started by adding 10 µL of human acetylcholinesterase (AChE). The absorbance was followed for 5 min and the inhibition curves were analysed by non-linear regression analysis to get the first order inhibition rate constant kStructural comparison and cartoon representations of protein structures were performed using PyMOL system . Root meFar-UV and near-UV CD spectra were recorded with JASCO J-715 spectropolarimeter according to the procedure described previously in the literature ,52 with Our results suggested a close relationship between dimerization, thermal stability and enzymatic activity for this class of enzymes. Also, a question on the mechanisms for the catalytic activity of PLL enzymes has emerged; if proper dimeric conformation is crucial for the catalytic activity of PLL enzymes, dimer or interface of monomers could provide a driving force for the activity. In other words, formation of dimer might be indispensable for this class of enzyme to retain their activity. This has highlighted the importance of mutagenesis approach that considers not only the active site of single monomer, but also effect on dimerization.


N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1–6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines . However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.The ongoing development of new production methods may lead to the commercialization of  N-acetylglucosamine, which is the second most abundant polysaccharide in nature after cellulose, and it is commonly found in crustacean shells, insect cuticles, and fungal cell walls . The RAW264.7 cells (2 × 105 cells/mL) were plated on 96-well plates (100 µL/well) and cultured for 24 h. Then, cells were incubated for 24 h in the presence of 100 µg/mL COS, NACOS, or 1 µg/mL LPS. The LPS-treated group of RAW264.7 cells were pretreated with 1 × PBS, 100 µg/mL of COS, or NACOS for 24 h in serum-free DMEM medium, followed by co-culture with 1 µg/mL LPS (positive control) for an additional 24 h. Then, a volume of 50 µL of Griess reagent was added to the culture supernatants. The absorbance was read at 540 nm, and the NO concentration was calculated using a standard curve of NaNO2.The NO content of the cell culture medium was measured, using the Griess reagent [1% sulfanilamide and 0.1% 5 cells/mL) were plated on 96-well plates (100 µL/well) and cultured for 24 h. Cells were incubated for 4 h in the presence of 100 µg/mL COS, or NACOS, or 1 µg/mL LPS. Moreover, the LPS-treated group of RAW264.7 cells were pretreated with 1 × PBS, or 100 µg/mL of COS or NACOS for 24 h, followed by co-culture with 1 µg/mL LPS in each well for 4 h. The cells were further incubated with 200 µL of 1 × PBS, containing 0.1% of NBT  and 2 µg/mL phorbol 12-myristate 13-acetate (PMA) at 37 °C for 0.5 h. The reaction was terminated with 80 µL of 70% methanol. Then, the cells were air-dried, and the blue formazan produced was dissolved in 120 µL of 2M KOH and 140 µL of DMSO. The absorbance was measured by a spectrophotometer at 620 nm, with KOH/DMSO as the blank.The ROS production of RAW264.7 macrophages was determined using the nitro blue tetrazolium (NBT) reduction test. The RAW264.7 cells (2 × 105 cells/ell) were seeded onto glass coverslips in a 12-well plate and allowed to adhere to the coverslips for 12 h. Then, cells were incubated for 24 h in the presence of 100 µg/mL COS, NACOS, or 1 µg/mL LPS. Moreover, the LPS-treated group of RAW264.7 cells were pretreated with 1 × PBS, or 100 µg/mL of COS or NACOS for 24 h, followed by co-culture with 1 µg/mL LPS in each well for 4 h. Then, cells were incubated with FluoSpheres™ Polystyrene Microspheres  at a multiplicity of infection of 5 at 37 °C for 1.5 h. Cells were washed three times with PBS to remove the undevoured beads. Then, the cells were analyzed with a flow cytometer . The extent of phagocytosis was analyzed, using a FlowJo system. The phagocytosis rate was calculated as the percentage of counts of cells engulfing microspheres in the gate of higher florescence value (counts of right peak) to all cell counts (sums of none-engulfing cells in left peak and engulfing cells in the right peak) [The RAW264.7 macrophage cells (4 × 10ht peak) .−ΔΔCt method. The primers for qPCR are listed in The RAW264.7 macrophage cells were treated with 100 µg/mL COS or NACOS, or 1 µg/mL LPS for 8 h, while the LPS-treated group of RAW264.7 cells was pretreated with 1 × PBS and 100 µg/mL of COS or NACOS for 24 h, followed by co-culture with 1 µg/mL LPS for 8 h. Total RNA was extracted from RAW264.7 macrophages using the TRIZOL reagent , according to the manufacturer’s protocol. Real-time PCR was performed on a LightCycler96 real-time system , using the SYBR Green Real-time PCR Master Mix , according to manufacturer’s instructions. Expression of the target gene was normalized to the housekeeping gene, β-actin, and calculated using the 2g) at 4 °C for 10 min. The protein contents were measured with the BCA protein assay kit , using bovine serum as the standard.The RAW264.7 macrophage cells were treated with 100 µg/mL COS or NACOS, or 1 µg/mL LPS for 8 h, while the LPS-treated group of RAW264.7 cells were pretreated with 1 × PBS and 100 µg/mL of COS or NACOS for 24 h, followed by co-culture with 1 µg/mL LPS for 8 h. The cells were washed three times with cold PBS and lysed with nuclear and cytoplasmic extraction reagents . Then, the cells were subjected to centrifugation  and proteins were transferred onto a polyvinylidene difluoride membrane by electroblotting . After the membrane was blocked with 5% skim milk containing Tris-buffered saline  for 1 h at 37 °C, the blots were incubated with mouse monoclonal antibody anti-IκBα ; anti-p-IκBα ; anti-IL-1β ; anti-β-actin ; rabbit monoclonal antibody anti-NF-κB p65 ; anti-NF-κB p-p65 ; and anti-Lamin B1  overnight at 4 °C. Following incubation with an appropriate secondary antibody , and scanned. Protein expression was corrected depending on the amount of β-actin or Lamin B1 present in a given sample.t-test and one-way AVOVA, followed by the Tukey’s post hoc test. p-values < 0.05 were considered to represent a statistically significant difference.All data were expressed as the mean ± SD of triplicate samples and were representative of at least three separate experiments. Statistical comparisons were made using the Student’s


How atmMOB are able to sustain life on the low CH4 concentrations in air is unknown. Here, we show that during growth, with air as its only source for energy and carbon, the recently isolated atmospheric methane-oxidizer Methylocapsa gorgona MG08 (USCα) oxidizes three atmospheric energy sources: CH4, carbon monoxide (CO), and hydrogen (H2) to support growth. The cell-specific CH4 oxidation rate of M. gorgona MG08 was estimated at ~0.7 × 10−18 mol cell−1 h−1, which, together with the oxidation of CO and H2, supplies 0.38 kJ Cmol−1 h−1 during growth in air. This is seven times lower than previously assumed necessary to support bacterial maintenance. We conclude that atmospheric methane-oxidation is supported by a metabolic flexibility that enables the simultaneous harvest of CH4, H2 and CO from air, but the key characteristic of atmospheric CH4 oxidizing bacteria might be very low energy requirements. The second largest sink for atmospheric methane (CH CO: −137.16 kJ/mol, O2: 0 kJ/mol, CO2: −394.39 kJ/mol, H2: 0 kJ/mol, H2O: −237.13 kJ/mol, CH4: −50.6 kJ/mol. From the oxidation rates and free energy change of the reactions, we could estimate the amount of energy obtained per mol of biomass carbon per hour, applying the dry weight and carbon content of M. gorgona MG08 (see below) as previously shown , [2CO + O2 → 2CO2], [2H2 + O2 → 2H2O]. Based on the measured rates, we find that M. gorgona MG08 is able to conserve approximately 0.47 kJ Cmol h−1 from the combined oxidation of CH4, H2 and CO during growth in air at 20 °C  are three times lighter (0.88 × 10−13 gDW cell−1) than the lightest reported MOB [M. gorgona MG08 may reflect a strategy to reduce energy costs for growth. In contrast to our findings, the previously reported soil CH4 oxidation rates of 800 × 10−18 mol cell−1 h−1 [−1. In order to examine this contradiction and test the validity of our data, we repeated parts of our experiment  with shorter pre-incubation (4.5 months) to minimize growth stagnation and the possibility for the accumulation of dead cells on the floating filters.The energy yields per mol of CHona MG08 . Interesll−1 h−1  would pr4, H2 and CO uptake rates . Within this timeframe, the leakage of H2 and CH4 was negligible  during growth in air at 20 °C (−1) [M. gorgona MG08. Thus, we question whether the average maintenance requirement of bacteria [M. gorgona MG08 and other atmMOB.With these new numbers, we find that at 20 °C . This is−1 minimum requirement mentioned above could be achieved if approximately 14 million out of the ~100 million cells on a filter were active [4 concentration) were lower during growth in air, an energy yield of 2.8 kJ Cmol h−1 or higher, could be achieved. The reason is that the same amount of energy would be distributed on less cellular mass than assumed for our empirical estimate of 0.38 kJ Cmol h−1. Regardless, the rates of M. gorgona MG08 are substantially lower than those in high upland soils, where CH4 oxidation rates of up of 800 × 10−18 mol cell−1 h−1, almost 500 times faster than M. gorgona MG08, were measured. However, these were based on soil cell numbers estimated from DNA extractions and pmoA qPCR [−1) than previously believed (9.8 kJ Cmol h−1) [M. gorgona MG08 can sustain its slow growth at a limited energy budget.With our numbers, the 2.8 kJ Cmol hmoA qPCR . Thus, tmol h−1) . AccordiM. gorgona MG08 oxidizes the atmospheric trace gases CH4, CO and H2 to harvest energy for growth in air. The ability of M. gorgona MG08 to grow using air as its only source of energy and carbon relies not only on this metabolic flexibility, but also on its low energy requirements. Our findings suggest that a high CH4 affinity is not a prerequisite to live on atmospheric CH4.We conclude that


Panax species, are considered the main active ingredients responsible for various pharmacological activities. Herein, a new protopanaxatriol-type ginsenoside called “ginsenoside MT1” is described; it was accidentally found among the enzymatic conversion products of ginsenoside Re. Method: We analyzed the conversion mechanism and found that recombinant β-glucosidase (MT619) transglycosylated the outer rhamnopyranoside of Re at the C-6 position to glucopyranoside at C-20. The production of MT1 by trans-rhamnosylation was optimized and pure MT1 was obtained through various chromatographic processes. Results: The structure of MT1 was elucidated based on spectral data: (20S)-3β,6α,12β,20-tetrahydroxydammarene-20-O-[α-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside]. This dammarane-type triterpene saponin was confirmed as a novel compound. Conclusion: Based on the functions of ginsenosides with similar structures, we believe that this ginsenoside MT1 may have great potential in the development of nutraceutical, pharmaceutical or cosmeceutical products.Background: Ginsenosides, triterpene saponins of  GinseVarious methods have been reported for preparing minor ginsenosides with high conversion efficiency and few byproducts . Many en1, Rg1 and Rg2) was accidentally found using a novel recombinant glucosidase (MT619) [In our investigation into the enzymatic conversion of major ginsenosides, a new transformation product of ginsenoside Re that is different from the other hydrolyzing products  . This co1 and Re) used in the present study were purchased from Sigma Co., Ltd. . F1, Rg2(S), Rh1(S) and PPT were prepared as described in our previous study [The standard forms of various ginsenosides  of 0.6, at which point protein expression was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The bacterial cells were incubated further for 24 h at 18 °C and then harvested by centrifugation at 4000 rpm for 15 min. The cells were washed with 50 mM sodium phosphate and then resuspended in 50 mM sodium phosphate and 1% Triton X-100 (pH 7.0). The cells were disrupted by ultrasonication . Intact cells and debris were removed by centrifugation at 13,000 rpm for 10 min to obtain a crude cell extract. His-tagged MT619 protein was purified by a HisTrap column . The 100 mM imidazole eluted protein was further purified by DEAE-cellulose DE-52 chromatography . Purified MT619 was used to examine its MT1 production efficiency by reacting with ginsenoside Re  in a shaking incubator at 37 °C for 24 h. The ginsenosides in the samples were extracted with an equal volume of butanol and identified by thin layer chromatography (TLC).Recombinant MT619 was expressed in eviously . BrieflyGinsenoside MT1 production was evaluated using crude MT619 cell extracts. To determine the optimal concentration of selected substrates for the biotransformation reaction, cells were mixed with an equal volume of a substrate at 3.0–22.5 mg/mL at 37 °C. Samples were then withdrawn at regular intervals and analyzed by TLC.g for 15 min and the supernatant was loaded into a column packed with HP20 resin (120 g) . One liter of water was used to remove unbound hydrophilic compounds and free sugar molecules, and the absorbed ginsenosides were eluted using three bed volumes of 95% ethanol. The eluted ethanol was evaporated in vacuo to produce dry powders.Production was scaled up to 1 L and a final concentration of 6.0 mg/mL substrate (ginsenoside Re). The transformation was performed by adding 300 mL of crude cell lysate in a shaking incubator at 200 rpm and 37 °C. After incubating for 24 h, the mixture was centrifuged at 4000 × g for 20 min. After filtering with a syringe filter (0.2 µm), the dissolved sample was subjected to liquid chromatography  and eluted with methanol-water  to yield 13 fractions. The elution was fractionated every 100 mL, and the fractions containing MT1 were collected for purification.Chromatographic pre-purification was performed with an LC-Forte/R system . One gram of the crude MT1 was dissolved in 20 mL of 10% methanol and centrifuged at 4000 × The collected MT1 was further purified using RPHPLC . RPHPLC was performed using a pre-packed column  purchased from Japan Analytical Industry Co. (Japan). The mobile phase was 50% acetonitrile; the flow rate was 7.0 mL/min. The MT1 solution was prepared by dissolving a sufficient quantity of the collected MT1 in 50% acetonitrile to give a final concentration of 35 mg/mL; 10 mL of the MT1 solution was loaded for purification.HPLC analysis of samples in the present study was performed using an Agilent 1260 Infinity HPLC system . Ginsenosides were separated on an YMC ODS C18 column  with a guard column . The gradient elution system consisted of water (A) and acetonitrile (B), and used the following gradient program: 0 → 10 min, 20% B; 10 → 40 min, 20 → 32% B; 40 → 48 min, 32 → 42% B; 48 → 60 min, 42 → 45% B; 60 → 83 min, 45 → 75% B; 83 → 85 min, 75 → 100% B; 85 → 95 min, 100% B; 95 → 95.01 min, 100 → 20% B; 95.01 → 100 min, 20% B. The detection wavelength was set to 203 nm and the flow rate was 1.6 mL/min.1H) and 175 MHz (13C), with chemical shifts given in ppm. Samples were separated with an Acquity UHPLC BEH C18 column  at room temperature. The mobile phases comprised deionized water containing 0.2% (v/v) formic acid (A) and acetonitrile (B). Quantitative analysis of the novel compound was performed using an LTQ-Orbitrap XL mass spectrometer  equipped with an ESI source  in negative ion mode with a mass range of m/z 100–1500 Da. Optimal conditions were employed as follows: capillary voltage 20 V, capillary temperature 350 °C, spray voltage 3.5 kV and tube lens voltage 110 V.NMR spectra were obtained using a Bruker AVANCE III 700 NMR spectrometer  at 700 MHz . TLC was conducted using 60F254 silica gel plates  and CHCl3-CH3OH-H2O  as the developing solution. The results were visualized with 10% H2SO4 by heating at 110 °C for 5 min.The purified compound (10 mg) was heated with 5% HCl mixture for 2 h at 70 °C. The residues from filtration and standard sugars were compared through cellulose TLC , which had a different Rf value from those of ginsenoside Rg1 and Rg2, was identified from Re reaction mixtures (2(S) was also hydrolyzed by MT619 into PPT, but MT1 was not reported. Interestingly, MT1 was only found in the Rg2 mixture containing F1  mixtures .Optimal conditions for the biotransformation of Re into MT1 were determined by examining the substrate concentrations in the reaction mixture. Enzyme reactions were performed using of ginsenoside Re, which is one of the major ginsenosides in ginseng ,24. The Six grams of Re in 1.0 L of phosphate buffer (pH 7.0) was reacted with cell lysates containing MT619. Re was completely converted 24 h after adding MT619 B. To remRecycling preparative HPLC, which can enhance the separation of compounds by recycling the effluent sample many times over the column without increasing the length of the chromatographic column, was used for further purification. MT1 was purified from the product mixture with a preparative ODS column by RPHPLC. Dry MT1 mixture (300 mg) was loaded onto the RPHPLC column for separation. MT1 was baseline-resolved after three effective columns . The pur42H72O13 (2(S) and neohesperidin  and 175 MHz (13C) with chemical shifts given in ppm for atomic force microscopy (AFM) to determine the chemical structure of MT1 (1H-NMR spectrum showed one olefinic (δH 5.28), two anomeric (δ 5.25 and 6.61) protons and a methyl proton signal (δ 1.83) like in L-rhamnopyranoside. Coupling constants suggested the configuration of the anomeric positions to be α-and-β form for the anomeric proton signals in the 1H-NMR spectrum. The 13C-NMR signals of the sugar were observed as one hemiacetal (δC 96.7), four oxygenated methines  and one oxygenated methylene (δC 62.95), indicating that the sugar is b-glucopyranose.Structural characterization was performed by a Bruker AVANCE III 700 NMR spectrometer operating at 700 MHz  correlations between the proton signal at δ 5.25 (H-1”) and the carbon signal at δ 84.0 (C-20). A large downfield shift in the C-2” (δ 76.6) was observed for the inner β-D-glucopyranosyl moiety at C-20 of the aglycone, which showed that the C-1’’’ (δ 6.61) in the terminal α-L-rhamnopyranosyl moiety is linked to the inner β-D-glucopyranosyl moiety at C-20 [1. When MT619 reacted with Rg2, the rhamnosyl transformation could only occur with the existence of the attached glucopyranoside at C-20 positions such as F1. The mechanisms of rhamnose transformation by MT619 warrant further exploration.Based on the above experimental results, the conversion pathways were deduced as shown in Phanerochaete chrysosporium can hydrolyze and transglycosylate glucopyranoside from laminarioligosaccharides [Aspergiluus niger strain ASKU28 also showed transglucosidase activity against various substrates [Many glycoside hydrolases have been shown to possess transglycosylation capabilities in addition to hydrolytic activity . In subscharides . β-glucobstrates . MT619 h1((20S)-3β,6α,12β-trihydroxydammar-24ene-(20-O-β-D-glucopyranosyl-(1→2)-α-D-glucopyranoside)) was synthesized using a cyclodextrin glucanotransferase [1 [1 a-glycosylated ginsenosides  linked at C-6 and C-20 positions of aglycones have been synthesized using rice seed a-glucosidases [Some glycosylated ginsenoside derivatives from various natural ginsenosides have been synthesized using enzymatic methods. A novel α-glycosylated ginsenoside Fnsferase ; ginsenoerase [1 . In addiosidases , althoug18 . This unknown compound can be efficiently produced from abundant ginsenoside Re using MT619. Various studies are on-going by our group to investigate its pharmacological effects. We expect potential commercial uses of MT1 in nutraceutical, pharmaceutical or cosmeceutical areas.In summary, this paper describes characterization and mass production of a new dammarane-type triterpene saponin from enzymatic converted products. The trans-rhamnosyl activity of MT619 against ginsenoside Re to F


II(ɳ6-p-cym)(PhAzPy-NMe2)I]+ (FY26) exhibits promising in vitro antitumour activity against mouse hepatocarcinoma Hepa1–6 and other mouse or human cancer cell lines. Here, we drastically enhance water solubility of FY26 through the replacement of the PF6− counter-anion with chloride using a novel synthesis method. FY26⋅PF6 and FY26⋅Cl displayed similar in vitro cytotoxicity in two cancer cell models. We then show the moderate and late anticancer efficacy of FY26⋅PF6 and FY26⋅Cl in a subcutaneous murine hepatocarcinoma mouse model. Both efficacy and tolerability varied according to FY26 circadian dosing time in hepatocarcinoma tumour-bearing mice. Tumour and liver uptake of the drug were determined over 48 h following FY26⋅Cl administration at Zeitgeber time 6 (ZT6), when the drug is least toxic (in the middle of the light span when mice are resting). Our studies suggest the need to administer protracted low doses of FY26 at ZT6 in order to optimize its delivery schedule, for example through the use of chrono-releasing nanoparticles.The organo-osmium complex [Os  We find that the efficacy and tolerability of FY26⋅PF6 or FY26⋅Cl are dependent on the time of administration, consistent with a role for the circadian clock in controlling expression of genes involved in glutathione synthesis.Metallodrugs differ from most organic drugs in that they are prodrugs, which undergo transformation by ligand substitution and redox reactions before reaching target sites. Here, we study the 2 and compounds of heavier congener osmium are being explored.4 Although in the same periodic group, and often possessing complexes of similar composition , the reactivity of ruthenium and osmium complexes can differ markedly. Notably, organo-Os(II) arene complexes hydrolyse much more slowly and bound water ligands are more acidic.5There is a need for anticancer drugs with new mechanisms of action to complement platinum drugs in the clinic, reduce side effects, and overcome emerging platinum resistance. Complexes of ruthenium have been investigated in clinical trials,7 For instance, dosing an anticancer medication at a specific time with reference to circadian rhythms can increase or decrease both efficacy and tolerability.8–10 As demonstrated for 28 anticancer agents belonging to various classes, the administration of a drug at the circadian time when it is best tolerated often achieves best antitumour activity.10 Here, we sought the relevance of this chronopharmacological principle along preclinical developmental stages of organo-Os complexes.Numerous experimental and clinical studies have highlighted the relevance of circadian timing in drug administration for the optimization of pharmacological effects.in vitro against a variety of cancer cell lines,12 and also in vivo,13 in contrast to less-active organo-Os(II) drugs with N,N-chelated diamine or N,O-chelated picolinate ligands, which rely primarily on the dissociation of labile ligands followed by DNA binding.15 Iodido Os(II) PhAzPy complexes are considerably more stable in aqueous media and exhibit a mechanism of activity involving production of intracellular reactive oxygen species (ROS). These inert complexes are activated in cancer cells by attack of glutathione on the azo bond of the chelated PhAzPy ligand.17 A key complex, FY26⋅PF6 ([OsII(ɳ6-p-cym)(PhAzPy-NMe2)I]PF6), is on average 49 times more active than cisplatin in the 809 cancer cell line screen of the Sanger Institute,18 and capable of delaying the growth of HCT-116 human colon cancer xenografts in mice.13 Furthermore, it is up to 64 times more selective towards A2780 ovarian cancer cells over MRC-5 fibroblasts when applied synergistically with l-buthionine sulfoximine (l-BSO).19 The structure of FY26 and its biomolecular mechanism of action are shown in Fig. Organometallic half-sandwich Os(II) arene complexes with phenylazopyridine (PhAzPy) bidentate ligands show promising anticancer activity In vitro experiments have also demonstrated time-dependent efficacy of FY26⋅PF6 against both mouse Hepa1–6 hepatocarcinoma and human HCT-116 colon carcinoma in cell cultures.21 More specifically, the IC50 of FY26⋅PF6 varied by 40% according to the timing of in vitro drug exposure of cultured Hepa1–6 cells, following the synchronization of their circadian clocks with a 24-h periodic temperature cycle.22 The least toxic timing for FY26⋅PF6 administration was near the minimum of the sinusoidal temperature cycle that synchronizes the genetic circadian clocks of the cells in the Petri dishes over the 24 h. FY26⋅PF6 chronopharmacology mechanisms depend upon clock gene Bmal1 proficiency, and involve circadian-dependent FY26 uptake and possibly efflux.2 Moreover, the toxicity of FY26⋅PF6 also varies according to dosing time in mice.22 The best-tolerated time for FY26 in mice following dosing is near the middle of the light span, which corresponds to the low point in their endogenous circadian rhythm in core body temperature. Strikingly, the circadian rhythm in FY26⋅PF6 toxicity for Hepa1–6 cells and for mice differs from those in oxaliplatin toxicity by several hours, hence further highlighting the pharmacological differences between these organo-osmium complexes and platinum metallodrugs on the temporal timescale.22Here, we aimed to optimize the chemical solubility of FY26 prior to the determination of FY26 efficacy and tolerability in Hepa1–6 tumour-bearing mice. We further obtained insight into its tissue uptake, so as to optimize its dosing schedule, in terms of both the frequency of administration and circadian timing.6 was synthesized and characterized as previously reported12, however, it was poorly water-soluble. We previously reported that the anion  instead of PF6. Furthermore, the route by which FY26 is synthesized was improved upon, now involving a two-step one-pot synthesis from [Os(η6-p-cym)Cl2]2. FY26 was first isolated as an iodide salt (FY26⋅I), which is an important purification step prior to anion exchange with chloride. The anion exchange was conducted by passing FY26⋅I through Dowex® 1X4 chloride form resin, yielding FY26⋅Cl.FY26⋅PF6-p-cym)Cl2]2  was dissolved in methanol (50 ml) and heated to 80°C. A solution of potassium iodide  in water (50 ml) was added and the mixture stirred for 5 min. A solution of N,N-dimethyl-4-(2-pyridylazo)aniline  in methanol (50 ml) was added dropwise and the mixture was stirred for 1 h at 80°C, and left stirring for 18 h at ambient temperature. The majority of methanol was removed under reduced pressure using a rotavap and the product was extracted with dichloromethane (100 ml), and washed with water (100 ml). More product was extracted from the aqueous layer with dichloromethane washings (4 × 50 ml). The combined dichloromethane extracts were vacuum filtered to remove a black impurity, and the filtrate was dried under reduced pressure. The solids were fully redissolved in a mixture of ethanol (200 ml) and dichloromethane (50 ml). The volume of the solution was reduced using a rotavap to ∼40 ml and a precipitate formed, and then the mixture was placed in a freezer (−20°C) overnight. The intermediate product (FY26⋅I) was collected via vacuum filtration and washed with ice-cold ethanol (∼10 ml), and then diethyl ether (6 × 50 ml). The washes were repeated once more. FY26 was collected, analysed by high-performance liquid chromatography , and dissolved in methanol (100–150 ml). reporter constructs within the C5SYS ERASYSBIO+ project (FP7). Mouse Hepa1–6-Per2-luc cells were cultured in 15-cm Petri dishes until they reached 80% confluency. On the day of tumour inoculation, the cells were trypsinized and resuspended at a concentration of 106 cells/100 µl in PBS solution alone (Experiments 1 and 2) or with 1:1 MatriGel™  (Experiments 3–5) and kept on ice until injected.Mouse hepatocarcinoma /2, where L is the larger and W the smaller perpendicular diameter, and 1 cm3 corresponds to 1 g. Mice with an estimated tumour weight exceeding 10% of their body weight or those in poor general condition or loosing >15% of their starting body weight were euthanized, in compliance with our Home Office Project Licence specifications approved by the Animals in Science Committee and Animal Welfare and Ethical Review Body.Mean daily tumour weight per mouse was calculated as the average volume of the right and left flank tumours according to  with vehicle (controls), or 40, 60, or 80 mg/kg/injection of FY26⋅PF6 at ZT6 (in the middle of the light span) every other day for 12 days  at ZT6 or at ZT18 (four controls and six treated mice per ZT) every other day for 11 days .Experiment 2 aimed at assessing whether FY26⋅PFExperiment 3 aimed at assessing the efficacy and tolerability of the more soluble formulation of FY26⋅Cl in Hepa1–6-bearing C57Bl/6 mice. The mice were randomized to receive vehicle or FY26⋅Cl, at a cumulative dose of 60 mg/kg. Treated mice received ip injections of 20 mg/kg of FY26⋅Cl at ZT6 12, 14, and 17 days after tumour inoculation. Control mice received ip vehicle on the same days.Experiment 4 aimed at confirming efficacy and optimizing timing and scheduling of the same cumulative dose of 60 mg/kg of FY26⋅Cl. Mice were randomized to receive either vehicle or FY26⋅Cl as four injections of 15 mg/kg at ZT6 or ZT18 on days 11, 14, 17, and 21.Experiment 5 determined FY26⋅Cl uptake in mouse tumour and liver. Hepa1–6-bearing mice were randomly assigned to either treatment or control groups. They received a single ip injection of 5, 10, or 20 mg/kg of FY26⋅Cl or 0.1 M PBS (vehicle) at ZT6 , 5% DMSO  and 94% of 0.9% saline  under laminar flow. FY26⋅PF6 concentration was confirmed with ICP-MS. For Experiments 3–5, using FY26⋅Cl, a stock solution of 50 mg/ml FY26⋅Cl was prepared in 0.9% saline on the day of injection and diluted as required; the vehicle consisted of 0.9% saline.For Experiments 1 and 2, FY26⋅PF2. Once thawed, ∼90–110 mg of homogenized tissue was pipetted into microwave reaction vessels (10 ml), and tetramethylammonium hydroxide  was added. Tumour tissue was not homogenized and instead digested as whole tissue. It was weighed directly into microwave vials and TMAH was added . The samples were digested via microwave irradiation  at 200°C for 10 min. Digested samples were diluted in one part DDIW, and then centrifuged at 9000 rpm for 10 min at 20°C. The final analytical samples were prepared by a further 1 in 12.5 dilution in DDIW , ensuring a final TMAH concentration of 1% and total dissolved solids <0.1% and the total dissolved solids were <0.1%. TMAH digestion was selected to circumvent the formation of volatile Os species with nitric acid.Liver tissue was homogenized by manual mechanical grinding (pestle and mortar) while frozen under liquid N189Os in gas (He) and no-gas modes, and samples were referenced internally to 100Ru (10 ppb). A concentration calibration curve was produced by serial dilutions of an osmium standard (1000 ppm) in 1% TMAH: 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 ppb.Analyses of digested tissue samples were carried out on an Agilent Technologies ICP-MS 7900 spectrometer in a basic aqueous matrix of TMAH . The osmium concentrations were determined via isotopic detection of Body weight data were expressed as % body weight measured on the first treatment day. Tumour weights for individually treated mice were expressed as % of the median control values, in order to determine % inhibition. Mean and standard error or median and interquartiles were computed for the different treatment groups and experimental conditions, i.e. drug timing and day of assessment. Groups were compared using parametric or non-parametric ANOVAs, respectively.6, I, or Cl) were determined in DDIW. Their solubilities were measured as 0.0201 (±0.0007), 1.29 (±0.01), and 215 (±10) mM for X = PF6, I, and Cl, respectively. A dramatic 10,448-fold increase in solubility is observed by switching the counter-anion from PF6 to Cl . Furthermore, FY26 has a chiral metal centre and exists as a racemic mixture of two enantiomers as shown by its X-ray crystal structure.25 As previously reported for FY26⋅PF6,18 FY26⋅Cl similarly did not induce apoptosis on exposure in A2780 cells but did induce an increase in ROS (data not shown). Herein, the enantiomers were separated via chiral HPLC and individually tested against A2780 ovarian and A549 lung cancer cells .22In Experiments 1 and 2, tumours grew exponentially up to a median value of ∼1.2 and ∼1 g, respectively, which was reached 14 days after inoculation. In Experiment 1, a dose response was documented for tumour inhibition, with efficacy ranking as highest for the dose of 80 mg/kg/injection, intermediate for that of 40 mg/kg/injection, and least for 60 mg/kg/injection.6 dose, with a slightly better efficacy at ZT6  or for that of Experiment 3 versus Experiment 4 (P = 0.671), and no interaction term was validated, with two-way ANOVA. Large interindividual variations were found, however, since the average tumour weight that was measured in each mouse varied by >10-fold in both control and treatment groups of each experiment.The distributions of mean tumour weights per mouse were similar in control and treatment groups both within and between experiments before FY26 treatment in Experiments 3 and 4. Indeed, no statistically significant difference was found for comparisons of treatment versus control groups (nuFoxn1 (Experiments 1 and 2). Spontaneous partial tumour regressions and regrowth were noticed. Tumours tended to be smaller in the treated mice throughout each experiment, and a clear antitumour effect of FY26⋅Cl was demonstrated 20 and 23 days after tumour inoculation in Experiments 3 and 4, respectively . Thus, at experiment completion, the median percentage of FY26⋅Cl-induced tumour inhibition relative to controls was 76.5% in Experiment 3, where the drug was given at ZT6 only, and 42.6% in Experiment 4 irrespective of dosing time. At the end of Experiment 4, the percentage inhibition relative to control was 12.5% in the mice dosed with FY26⋅Cl at ZT6 and 66% in those treated at ZT18   as compared to the CD1-P = 0.0012). Body-weight loss, however, did not correlate with other signs of physical condition that triggered humane endpoints.Thus, at study completion, 13/20 controls in Experiment 3 and 10/10 controls in Experiment 4 were alive, representing an overall rate of 23/30 controls for both experiments (76.7%). Seven control mice (23.3%) had to be euthanized due to tumour progression. In contrast, severe clinical toxicity signs were observed that mandated euthanizing nearly half of the treated mice for ethical reasons. As a result, 8/16 (50%) and 9/20 treated mice (45%) were alive at completion of Experiments 3 and 4, respectively. In Experiment 4, overall survival was best following FY26 dosing at ZT6 as compared to ZT18 with respective survival rates of 60% versus 30% , as well as for circadian timing effects .Thus, the body weight changes showed a significant difference between the treatment and the control group after the third dose in each experiment. After a cumulative dose of 60 mg/kg of FY26⋅Cl, mice lost ∼10% (±3.2%) of their body weight before treatment. Body-weight loss from FY26 dosing was clearly worse at ZT18 as compared to ZT6 .6 anions have high crystal packing energies, and we previously reported that solubility can be improved up to 45-fold by switching to CF3SO3 as a counter anion.23 Here, we show that FY26⋅X solubility is vastly improved 10,448-fold by switching to a chloride anion. Such drastic solubility improvements eliminate the need for DMSO in biological assays and improve viability for clinical use. Furthermore, since the active component is the organo-osmium cation, the change in anion did not significantly alter its pharmacological activity.Organo-osmium anticancer complexes with PFII(ɳ6-p-cym)(PhAzPy-NMe2)I]+), a pseudo-octahedral complex in which the arene p-cymene occupies three coordination positions, the phenylazopyridine ligand is chelated, and a strongly bound iodide ligand completes the coordination sphere.28Among other precious metals, complexes of Os(II), also a third row transition metal, and isoelectronic with Pt(IV), show promise. Here, we have studied the organo-osmium(II) half-sandwich complex FY26 .37 Our study suggests that treatment schedules should indeed aim to reduce the exposure of healthy tissues to FY26⋅Cl through drug dosing at ZT6, because (i) a significant tumour drug uptake was demonstrated at this time, (ii) a significant tumour inhibition was achieved in mice treated at this time with the more dose-intensive schedule (Experiment 3), and (iii) the optimization of drug tolerability was crucial for outcomes (Experiment 4).In spite of the larger body weight loss, the mice receiving FY26⋅Cl at ZT18 had a larger tumour-weight reduction as compared to those injected at ZT6. The apparent increase in antitumour efficacy following dosing at the circadian time of poor tolerability strikingly differs from prior findings with other anticancer agents, including cisplatin and oxaliplatin. Thus, the dosing time of best efficacy coincided with that of best tolerability for 28 anticancer drugs.6 are least toxic to the mice, following dosing near the middle of their resting span, during daytime. The circadian pattern in the FY26⋅Cl tolerability that was found here was similar to that demonstrated earlier for the less soluble FY26⋅PF6 formulation. The circadian optimization of tolerability appeared potentially useful, because FY26-induced antitumour efficacy required treatment with repeated administrations. Thus, good tolerability would favour compliance to the subchronic treatment that was needed to achieve efficacy. Since the tumour drug levels remained similar at 48 h post-dose, irrespective of dose level, we propose to enhance treatment efficacy through the daily delivery of low dose levels (i.e. 5–10 mg/kg) at ZT6, a high dose intensity schedule that could be automatically delivered using ‘intelligent’ nanoparticles.Here, we have demonstrated that FY26⋅Cl and FY26⋅PF


Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes. Human 2′-5′-oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that form an important component of the innate immune system. The OAS family consists of different OAS enzymes: OAS1, OAS2, and OAS3. OAS enzymes are activated upon interaction with viral double-stranded RNAs and synthesize 2′-5′ linked oligoadenylate chains from ATP that further activate ribonuclease L, ultimately restricting viral propagation. We present the first, to our knowledge, low-resolution structural model of OAS2 dimer and confirm dimer formation in a cellular background. Our work provides insights into the recognition of double-stranded RNA of different lengths by OAS enzymes, critical for the innate immune response.β (The 2′-5′-oligoadenylate synthetases (OASs) are interferon-induced proteins that catalyze the synthesis of 2′-5′-linked oligomers (2-5A) upon interaction with double-stranded RNA (dsRNA) and are often triggered by viral infection . 2-5A caβ . Three aβ . Mutatioβ . In OAS1β . OAS2 coβ . OAS3 haβ .OAS enzymes are present in most mammalian cells and tissues ,17 and hdsRNA interacts with a channel of positively charged residues on the face opposite to the OAS active site ,27. OAS1Oligomerization of OAS enzymes and/or the orientation of individual OAS domains has been hypothesized to play a role in enzymatic activity. OAS1 forms monomer, dimers, and tetramers , whereasAlthough the high-resolution structures of OAS1 and domain I of OAS3 have provided important insights into the recognition of dsRNA, the importance of OAS self-association has not been as extensively studied ,27,29. SHindIII, purified using a Genejet PCR purification kit , and ligated using T4 DNA ligase (Thermo Fisher Scientific) into predigested pCDNA3 vector. pCDNA3 vector containing CAFAKA was transformed into NEB Turbo competent E. coli  using the manufacturer’s protocol. Maxi-Preps (Thermo Fisher Scientific) were performed to obtain plasmid DNA.Protein expression in HEK293T cells and subsequent purification of FLAG-tagged OAS2 (FLAG-OAS2) and His-tagged OAS2 (HIS-OAS2) were performed as described previously , with thCatalytically inactive aspartic acid mutant D481A  was constructed by PCR from FLAG-OAS2 in pCDNA3 using an overlapping forward primer (5′-TCCAAAGTCCTCAACGAAAGTGTCAGCTTTGCCGTGCTTCC TGCCTTTAATGCACTGGGTCAG-3′) and reverse primer (5′-CTGACCCAGTGCATTAAAGGCAGG AAGCACGGCAAAGCTGACACTTTCGTTGAGGACTTTGGA-3′). After PCR, the parent plasmid was digested with DpnI (Thermo Fisher Scientific) for 30 min at 37°C in the water bath. Cleanup of the PCR product and isolation of plasmid DNA were performed as described for CAFAKA. All plasmids were sequenced before transfecting HEK293T cells for protein expression. The expression and purification of the OAS2 mutants CAFAKA and D481A were performed as described previously .−1 cm−1) calculated with the ProtParam tool on ExPASy servers  in 20 mM HEPES (pH 7.2), 300 mM NaCl, 2 mM dithiothreitol (DTT), 0.1 mM EDTA, and 10% (v/v) glycerol. The eluted fractions were monitored by absorbance at 280 nm. Multiple fractions of individual peaks were combined and concentrated using Millipore concentrator filters . Protein purity was confirmed by sodium dodecyl sufate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined spectrophotometrically by absorbance at 280 nm using the extinction coefficient  produced by the catalysis of ATP by 2-5A formation as described previously . OAS2 was solubilized in a buffer containing 20 mM HEPES (pH 7.2), 300 mM NaCl, 2 mM DTT, 0.1 mM EDTA, and 10% (v/v) glycerol, whereas OAS1 was solubilized in buffer containing 20 mM Tris (pH 7.5), 100 mM NaCl, 1 mM DTT, and 10% (v/v) glycerol. 16 measurements were collected at each protein concentration examined, and the obtained hydrodynamic radii were extrapolated to infinite dilution to yield r0H.DLS measurements were performed at 20°C on a Nano-S DLS system  as described previously ,35. BefoμM) at a wavelength of 294 nm, and OAS1 concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 g/L (12–84.13 μM) for OAS1 at 20°C at a wavelength of 297 nm. Before data collection, OAS2 was dialyzed in 20 mM HEPES (pH 7.2), 300 mM NaCl, 0.1 mM EDTA, 2 mM DTT, and 10% (v/v) glycerol, and OAS1 was dialyzed in 20 mM Tris (pH 7.2), 100 mM NaCl, 1 mM DTT, and 10% (v/v) glycerol. 400 μL of each sample was loaded into a channel of the double-sector centerpiece. The rotor with samples were equilibrated to 20°C for ∼2 h under vacuum. Radial scans for OAS2 and OAS1 were collected every 10 and 11 min, respectively. One-dimensional distributions c(s) of the sedimentation coefficient (S) were calculated in SEDFIT (s), apparent molecular mass (M), and oligomerization (if any) at each concentration in SEDFIT  of 1.040600 g/cm3 and 1.003800 g/cm3, a buffer viscosity  of 0.014145 P and 0.010270 P, and partial specific volume 3/g for OAS2 and OAS1, respectively, to obtain s0C, w20° and M0 (Sedimentation velocity (SV) data were collected using a ProteomeLab XL-I analytical ultracentrifuge  with an An-50 Ti 8-place rotor, using absorbance optics at a rotor speeds of 30,000 and 25,000 revolutions per minute (rpm) for OAS2 and OAS1, respectively. We measured OAS2 concentrations of 0.16, 0.41, 0.57, and 1.33 g/L  distributions, were generated with GUSSI , distorted α-helix (helix-2), regular β strands (strand-1), distorted β strands (strand-2), turns (T), and unordered (U), devised by Sreerama et al. , 300 mM sodium fluoride, 0.1 mM EDTA, and 10% (v/v) glycerol, followed by data collection using a J-810 spectropolarimeter , and a 0.05 cm quartz cell . Samples and baseline buffer spectra were measured under a continuous scanning mode with a 2 nm data pitch and a scan speed of 5 nm/min with a response time of 2 s. The CD spectra presented are from the accumulation of four scans of OAS2 protein at 0.478 g/L with the buffer signal subtracted. CD data were deconvoluted using the online DICHROWEB webserver analysis tool , which pa et al. . CD clase PHYRE2  online wμL of purified samples was loaded onto a Shodex KW404 column , followed by SAXS data collection every 3 s as described previously  using an SEC-SAXS setup. 50 eviously . An in-leviously . The bufeviously , as desceviously ,49.rG) and study OAS2 homogeneity  were determined by calculating the pair-distance distribution P(r) function using the program GNOM (4RWN) by means of SWISS-MODEL, using the default web interface  function was performed using the approach outlined for OAS2. To evaluate whether the low-resolution structures obtained from peak 1 of OAS1 data agree with a dimeric structure of OAS1, we designed a dimeric model of OAS1 using the CLUSPRO program (4IG8) to obtain a dimer of OAS1. Similar to OAS2, we performed free docking of OAS1 using CLUSPRO and obtained ∼100 atomistic structures. Using the program CRYSOL  of polyethylenimine , and the volume was brought to 5 mL with serum-free DMEM. The transfection mixture was kept at room temperature for 20 min and then added to the cell culture plates dropwise. Recombinant pCDNA3 vector (Thermo Fisher Scientific) containing FLAG-OAS2 and HIS-OAS2 were used to transfect the cells either singly or in combination along with a negative control (empty vector). Cells were incubated at 37°C for 2 days, then harvested using 1 mL of cold phosphate-buffered saline 1× (PBS). Cells were pelleted by centrifugation at 1500 rpm for 5 min at 4°C, resuspended into 1 mL cold phosphate-buffered saline, and repelleted at 2500 rpm for 3 min at 4°C. To acquire the cytoplasmic fractions, the cells were resuspended in 275 μL of cytoplasmic lysis buffer (25 mM HEPES (pH 7.2), 5 mM KCl, 0.5 mM MgCl2, 0.5% octylphenoxy poly(ethyleneoxy) ethanol (IGEPAL), and 1× halt protease inhibitor cocktail, EDTA free). The suspension was end-over-end rotated for 5 min, and the insoluble material was pelleted by centrifugation for 5 min at 5000 rpm. The soluble cytoplasmic fraction was retained. The insoluble fraction was further mixed with 275 μL of nuclear lysis buffer (25 mM HEPES (pH 7.2), 350 mM NaCl, 0.01% IGEPAL, 10% w/v sucrose, and 1× halt protease inhibitor cocktail, EDTA free), vortexed, and passed through a 20 gauge needle in triplicate. The suspension was mixed end over end for 10 min and then centrifuged at 4°C for 10 min at 14,000 rpm. Soluble nuclear fractions of the cell lysate were then combined with retained soluble cytoplasmic fractions of the cell lysate. 50 μL of this lysate was set aside for SDS-PAGE gel (pre-IP) and Bradford assay to measure the protein concentration before the IP. The remaining lysate (500 μL) was divided into two 250 μL fractions into microfuge tubes and immunoprecipitation (IP) was performed in total IP buffer (25 mM HEPES (pH 7.2), 175 mM NaCl, 2.5 mM KCl, 0.25 mM MgCl2, 0.25% IGEPAL, 5% w/v sucrose, and 1× halt protease inhibitor cocktail, EDTA free). 2.5 μg of anti-HIS, monoclonal anti-FLAG antibody  was added to each tube, and 2.5 μg of IgG negative control antibody was added to the lysate where cells were transfected in combination and mixed end over end for 1 h at 4°C. In a 2 mL microfuge tube, 30 μL of protein A/G beads (Thermo Fisher Scientific) was equilibrated in total IP buffer three times for 10 min, then magnetically pelleted to the side wall of the microfuge tube. The buffer was aspirated, and the IP sample was added to the microfuge tube and incubated for 1 h at 4°C with end-over-end mixing. Protein A/G beads were magnetically pelleted, and 50 μL of supernatant was set aside for SDS-PAGE and Bradford assays to measure the protein concentration after IP. The beads were then washed by end-over-end mixing three times for 10 min with 500 μL of total IP buffer, followed by manual pipette mixing. The beads were magnetically pelleted again, the supernatant was aspirated, and the pellet was resuspended in 100 μL of 1× SDS load dye. The solution was heated at 95°C for 5 min, then centrifuged at 14,000 rpm for 5 min at room temperature. 10% SDS-PAGE gels were run at 220 V for 35 min, and Western blots were performed using 50 μg of pre-IP and post-IP and 8 μL of the IP sample. Mouse monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich  (Cat. # F1804), and mouse monoclonal anti-HIS antibodies were purchased from Millipore (Cat. # 05-949).For the coimmunoprecipitation assay, HEK293T cells were grown on 100 mm cell culture dishes (Thermo Fisher Scientific) in 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and grown to 80% confluency. The medium was replaced with fresh DMEM with 10% fetal bovine serum just before transfection. The transfection mixture was made by adding 30 A). Extrapolation of the plot of rH measured at each concentration to infinite dilution provided a value of 7.5 ± 0.26 nm (B), which is significantly larger than the reported value obtained for OAS1 (3.0 ± 0.3 nm) (−13 s). AUC peaks suggest a contribution from both dimer (dominant) and monomer (minor). We obtained an s0w20,  (S) of 4.9 ± 0.3S for OAS2 (C). The c(s) analysis shows a clear trend of an increase in the signal with increasing OAS2 concentration  . The plofor OAS2 C. The cn see C;  and the olecules . Based oolecules ,64, suggβ-strand-1 and 2, which form 7 and 6%, respectively, of the total protein. In addition, OAS2 contains 17% turns and 25% unordered structures (D). These values were consistent on analysis with both the CONTIN-LL  and PYRE2 (data not shown) methods.Next, we performed CD experiments on OAS2 to determine the secondary structure content of the protein. The net CD spectrum was deconvoluted using the CDSSTR method and presented features of a folded-globular protein . The data suggest that OAS2 consists of helix-1 and 2, constituting 27 and 19%, respectively, of the total protein, and ructures D. TheseA). The signal in the chromatogram is the integral of the ratio of the scattering intensity of the individual frame/the background intensity measured from the buffer. Individual scattering intensity profiles that provided uniform rG distribution and displayed monodispersed distribution were merged using the program PRIMUS (B). Guinier analysis of the merged data demonstrated a monodispersed preparation (inset to B). The merged data from B were also used to perform dimensionless Kratky analysis (plot of (I(q)/I(0)) × (q × rG)2 vs. q × rG) that is particularly useful in determining global protein folding. The plot presented in C suggests a Gaussian distribution and a well-defined maximum, confirming that OAS2 is a well-folded protein. For a globular-shaped protein in solution, a dimensionless Kratky plot displays a well-defined maximal value of 1.1 at q × rG = C, OAS2 does not adopt an extended conformation in solution.To obtain solution structures of OAS2, we collected data using a coupled SEC-SAXS setup in which SAXS data were collected every 3 s during elution from a size-exclusion column. The sample eluted as a predominant peak at ∼400 s A. The sm PRIMUS  Fig. 2A. The srG) and forward angle scattering (I0) using the Guinier approximation (P(r) analysis (which represents a histogram of the interelectron distances within the structure) (D), in agreement with the frictional ratio obtained from SV-AUC experiments. Globular proteins usually have a bell-shaped P(r) curve, whereas elongated proteins have an extended tail (P(r) analysis, we obtained the rG and Dmax of OAS2 as 42 ± 0.25 Å and 110 Å, respectively . Consistent with dimeric OAS2, a volume-based molecular weight of ∼165 kDa was obtained from the solution structure, nearly twice the mass of the sequence-derived molecular weight (78.78 kDa) of OAS2.We calculated the radius of gyration (ximation  Table 1rG and foructure) ,67 was pructure) D, in agded tail . Based oructures ,69,70. Atructure  . A representative example is presented in B. The atomistic models derived using CORAL suggested that although OAS2 forms a dimer in solution, the catalytically active domains (DII) are located across the central cavity as opposed to being side by side. In parallel, we utilized CLUSPRO , FLAG-OAS2 can be efficiently detected. FLAG-OAS2 can also be detected after immunoprecipitation with anti-HIS  only under conditions in which both FLAG-OAS and HIS-OAS2 are co-transfected into cells. This suggests self-association between at least two OAS2 protomers. Under all other control immunoprecipitation conditions examined , no FLAG-OAS2 was detected. The reciprocal experiments using anti-FLAG immunoprecipitation with anti-HIS detection by Western blot demonstrated similar results, again suggesting OAS2 self-association (B).To confirm OAS2 self-association in the context of a cellular background, we transfected HEK293T cells with combinations of empty pCDNA3 vector, FLAG-tagged OAS2, and HIS-tagged OAS2 and performed coimmunoprecipitation experiments. ociation B.FigureA, highlighting the three active-site aspartic acid residues with asterisks and the C-terminus CFK residues with arrows. OAS2, CAFAKA, and D481A were expressed and purified simultaneously . To determine whether the CAFAKA mutant impacts enzymatic activity, we used an established OAS activity assay (B). CD analysis of D481A suggests a well-folded protein consistent with wild-type features (data not shown). We then repeated the assay at a single time point over a range of protein concentrations (0–1.43 × 10−2 g/L) to assess the impact of low concentrations that may disrupt dimer formation on activity. As expected, D481A did not display any enzymatic activity, but OAS2 and the CAFAKA mutant were active even at low protein concentrations, suggesting that these residues are not sufficient for dimerization (C).To further probe the importance of dimerization in OAS2 activity, we expressed and purified OAS2 mutants from HEK293T cells. Previous studies indicated that mutation of three C-terminal amino acids in the catalytic domain of OAS1 or OAS2 (CAFAKA mutant) causes a complete loss of activity in OAS1 and OAS2 purified from bacterial and insect cells, respectively, potentially because of the prevention of OAS self-association ,31. Mutaaneously B, insetty assay ,72 that ty assay B. CD anrization C.Figurec(s) analysis showed an increase in signal intensity with increases in OAS1 concentration . We also calculated the molecular mass of both OAS1 monomer and dimer using SEDFIT, which yielded ∼44.9 and 91.6 kDa, respectively; a single frictional ratio of 1.4 in continuous c(s) distribution model was used, and monomer and dimer distribution curves were integrated into SEDFIT to determine the molar mass (see Rh distribution (B). The hydrodynamic radius (rH) extrapolated to infinite dilution was 3.83 ± 0.05 nm, a value consistent with a distribution containing mainly monomers (C).Our previous studies on OAS1 using SV-AUC at low concentrations (up to 0.8 g/L) suggested that OAS1 is monomeric in solution . In thistion see  and yield values . Howeverd values A. We almass see . DLS in ribution B. The hmonomers C.Figureθ) than the home source.Previously, we published a low-resolution structure of OAS1  based onA. For OAS1, we obtained four peaks at different time points (A). Data processing of the first  and the second  peak provided sufficient scattering signal such that we could obtain the rG and Dmax from both peaks. The raw scattering data obtained from peak-1 (blue) and peak-2 (pink) show different scattering patterns, suggesting that a unique envelope can be extracted from each (B). We also performed Guinier analysis (inset to B) for both peaks, and the linearity observed by fitting the low-q data suggests that both peaks are monodispersed. Subsequently, we performed the P(r) analysis to obtain the rG and Dmax values from both peaks. The P(r) analysis for peak-2 resembled the previously published data (C), including the rG (24.88 ± 0.02 Å) and Dmax (70.0 Å), indicating that peak-2 corresponds to a monomeric version of OAS1. Furthermore, similar to OAS2, we performed a dimensionless Kratky analysis. As presented in D, this analysis suggests that peak-2 of OAS1 adapts globular conformation in solution, whereas peak-1 does not. Because peak-1 represents a higher molecular weight than peak-2, we investigated whether it potentially represents a dimeric version of OAS1. The P(r) analysis for peak-1 is consistent with an extended tail, suggesting the shape should be more cylindrical or ellipsoidal (C). The Rg obtained from P(r) plots for peak-1 and peak-2 were 34.10 ± 0.10 Å and 24.88 ± 0.02 Å, respectively. The Dmax determined for peak-1 and peak-2 are 110 and 70 Å, respectively  is similar to the previously published model obtained from the in-house scattering data (B), further indicating that peak-1 could represent a dimeric conformation. To determine whether the OAS1 structure obtained from peak-1 data can accommodate two OAS1 monomers, we performed docking using CLUSPRO . Interestingly, CFK residues , active-site aspartic acid residues , and the RNA binding interface are all surface exposed (C).We calculated 13–20 structural models for both peak-1 and peak-2, which were then rotated, aligned, and filtered as above to obtain a representative shape. We obtained the ures see . The NSDures see A is siming data . The string data B, furth CLUSPRO . Next, w CLUSPRO  program USPRO C; . The χ-vy likely C. Inter exposed C.FigureB and C). The differences in the arrangement of monomeric units are because both methods employ different algorithms and restraints . Therefore, rather than presenting the results from one of the two methods, we are presenting data from both methods to demonstrate that both conformations are possible and that without experimental evidence of the high-resolution structure of OAS2 dimer bound to RNA, it is very difficult to select any of the conformations with confidence. Coimmunoprecipitation assays suggest that OAS2 does indeed self-associate in a cellular background, as previously suggested . Incorporating previous biochemical observations, we propose the DI of one OAS2 protomer is tightly bound to the DI of another OAS2 protomer. Similarly, DII is also bound to DII of another OAS2 protomer providing it an overall globular shape. We hypothesize that dsRNA interacts with both catalytically active domains of the OAS2 dimer and requires interaction with a minimum of four minor grooves of dsRNA provided by ∼35 bp dsRNA. In addition, we also suggest that OAS2 protomers have very high affinity for each other, as is shown by the immunoprecipitation assays and supported by a tight dissociation constant of 5.1 nM previously observed . This result is in contrast with Sarkar et al., who suggested that CAFAKA leads to complete loss of activity (The residues C668, F669, and K670 present on the C-terminal end of OAS2 have been previously suggested to be important for its activity and dimerization . We madeactivity . HoweverAlthough our results suggest OAS2 adopts a dimer conformation under a wide range of protein concentrations, we observed that OAS1 adopts a dimer only at higher concentrations well above those that would be observed physiologically and is a minor species relative to the monomer. However, there are a limited number of reports suggesting that OAS1 self-association may be mechanistically important. Wang et al. recently suggested mechanistic differences in OAS1 activation with longer dsRNAs, and their results questioned whether the crystal structure of OAS1 in complex with 18 bp dsRNA is representative of a fully activated form of OAS1 ,77. AlthIn conclusion, we have demonstrated the hydrodynamic parameters of OAS2 and obtained the first, to our knowledge, low-resolution structures of OAS2 dimer. In addition, a combination of biophysical tools has provided us structural information about in-solution structures, stability, and homogeneity of OAS enzymes. This information could potentially help us to better understand how OAS enzymes exist in cells and interact with other proteins and nucleic acids and provides us with a better understanding of the OAS role during viral infections.