D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α- Inulin-type fructans and fructooligosaccharides, which are composed of β-2→1) fructosylfructose linkages, are attracting attention as prebiotics in functional foods  fructosy, 4. NatuD-Fruf-1,2′:2,3′-β-D-Fruf) and DFA IV  have various beneficial effects on human health  from levan or inulin have been identified (https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_91) (Difructose dianhydrides (DFAs) are cyclic disaccharides consisting of 2 fructose units reciprocally linked at their reducing carbons. The disaccharides are generated by the caramelization of fructose-containing foods . Commercn health . An indun health , 12. In entified . DFA IV-entified  belongs entified . In contentified  and DFA entified . The proentified  and DFA entified  are consmily_91) .D-Fruf-(2→1)-D-Fru) was isolated from the fungus Aspergillus fumigatus -D-Glc -D-Glc , suggestBifidobacterium dentium isolated from human dental caries  that belongs to a domain of unknown function (DUF) 2961, and a set of putative ABC transporter proteins (A). To investigate the in vitro function of the BBDE_2040 protein, we heterologously produced the C-terminal His6-tagged BBDE_2040 protein in Escherichia coli and purified it to homogeneity. As the model substrates to study the function of BBDE_2040, 1-methyl α-D-fructofuranoside (D-Fruf-α-Me), 1-methyl α-D-arabinofuranoside (D-Araf-α-Me), p-nitrophenyl α-D-fructofuranoside (pNP-α-D-Fruf), and p-nitrophenyl α-D-arabinofuranoside (pNP-α-D-Araf) were synthesized. When D-Fruf-α-Me and D-Araf-α-Me were incubated with BBDE_2040, spots of D-fructose and D-arabinose appeared on TLC (B and C), showing that BBDE_2040 has α-D-fructofuranosidase/α-D-arabinofuranosidase activities. We designate the BBDE_2040 protein as α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium (αFFase1) in this study because the natural substrates of this enzyme are supposed to be α-D-fructofuranosyl compounds (described below). pNP-α-D-Fruf was unstable in aqueous solution (A and B), and the activity of αFFase1 was apparently lower for pNP-α-D-Fruf than for pNP-α-D-Araf (B). Therefore, pNP-α-D-Araf was used as the standard substrate for the enzymatic characterization. The optimal pH and apparent optimal temperature were 5.5 and 50 °C, respectively (A and B). The Km and kcat values toward pNP-α-D-Araf were 2.71 ± 0.21 mM and 127.5 ± 4.0 s−1, respectively, at pH 6.0 and 37 °C (C).In the genome of proteins A. To invd on TLC , B and Csolution , A and Bα-D-Araf B. Therefectively , A and Bnd 37 °C C.Figure 1H NMR  disappeared within 1 min. A signal of H-1 of α-D-Araf  first appeared as the major initial furanoside concomitant with a much weaker H-1 signal of β-D-Araf . The anomeric H-1 signals of pyranoses, such as α-D-Arap  and β-D-Arap , subsequently appeared and clearly increased as more stable forms because of mutarotation under the equilibrium conditions within 30 min. This result indicates that αFFase1 is an anomer-retaining GH.The stereochemistry of the glycosidic bond hydrolysis was monitored by 1H NMR  and S3. B. dentium colonizes the human oral cavity and intestine  analysis of the caramelized D-fructose prepared by heat treatment at 130 °C for 120 min. After incubation with αFFase1, two compounds with retention times at 6.7 and 11.5 min (indicated as peaks 1 and 2) decreased and two compounds at 3.5 and 10.5 min (peaks 3 and 4) increased. Similar peak shifts from peaks 1 and 2 to peaks 3 and 4 were observed for the incubation of the caramelized D-fructose and D-glucose mixture with αFFase1 (B). After the overnight reaction, the molar ratio of peaks 1 and 3 was 11.6:88.4. MALDI TOF-MS analysis of these peaks and their peracetylated samples suggested that peaks 1 and 3 were a fructose disaccharide and its anhydride, respectively -α/β-D-Fruf/p -α-D-Fruf form of Frupβ2,1Fru was converted to DHL II by the catalysis of αFFase1 (C).Because ntestine , we hypo αFFase1 . When pu αFFase1 B. After ectively . NMR anaβ-D-Frup  and S1.  αFFase1 C.Figure D-Fruf-1,2′:2,1′-β-D-Frup) and DFA I  are structurally similar, we presumed that the conversion from peak 2 to peak 4 of the caramelized sugar was that from inulobiose (β-D-Fruf-(2→1)-α-D-Fruf) to DFA I. When inulobiose was incubated with αFFase1, DFA I was produced (A and 4A). The reactions of αFFase1 between inulobiose and DFA I were confirmed by 1H NMR (A) in both directions. The 1H and 13C NMR chemical shift data of the DFA I sample produced from inulobiose (B) were identical to those previously reported (1JH3–H4 (2.4 Hz) and 13C chemical shifts of C3 at 82.1 ppm unambiguously indicated that α-Fruf structure was formed from inulobiose. When the reaction was monitored by HPAEC-PAD using inulobiose or DFA I as a substrate, the reactions reached an equilibrium ratio of DFA I-inulobiose = 89.1:10.9 (4B). Matsuyama and Tanaka also reported that the enzyme from A. fumigatus resulted in a similar equilibrium biased to DFA I , and there were no significant peak changes in the reaction of caramelized sugars other than those of the four peaks .αFFase1 did not catalyze any reactions when DFA III or inulin was used as a substrate B, and thur peaks A and S4.4KQ7, 36% sequence identity) from Bacteroides uniformis ATCC 8492 as a search model. The ligand-free structure was determined at 1.96 Å resolution . The two β-jelly roll domains correspond to CATH superfamily 2.60.120.1390 (protein of unknown function DUF2961)  . A Dali DUF2961)  of the wDUF2961) . SeveralD-fructose and D-arabinose were determined at a resolution of 1.76 and 1.87 Å, respectively (f and β-Araf were observed in similar conformations at the expected −1 subsite (B and C). The furanose ring of β-Fruf and β-Araf adopts E5 (P ∼ 35°) and 3T4 (P ∼ 54°) conformations, respectively . The sugar ligands are surrounded by Trp267, Glu270, Glu291, Asp292, Trp298, and Tyr187′ . Notably, Glu291 and Glu270 are positioned close to the anomeric C2 atom from the β and α configuration sides, respectively. Therefore, Glu291 and Glu270 are suggested to be nucleophile and acid/base catalyst residues, respectively.The structures of αFFase1 complexed with ectively . In the  subsite , B and Cectively , as analectively . These cectively , and a ms E5 P ∼ ° and 3T4D, green sphere), although the buffers used during protein purification and crystallization contained no divalent metal ions. The metal ion in the crystal structure was assigned as Ca2+ based on the analysis using the CheckMyMetal server . Treatment with excess amount of EDTA did not inhibit the activity of αFFase1 (A). The effects of divalent metal ions showed that Cu2+ and Zn2+ inhibited the activity, whereas other metal ions including Ca2+ did not (B).A metal ion was observed close to the active site . In addition to Trp267, Glu85′ and Lys147′ seemed to constitute the +1 subsite.Extensive attempts at cocrystallization and soaking experiments with DFA I and inulobiose using the WT enzyme and mutants at the putative catalytic residues (Glu270 and Glu291) did not reveal electron densities in the active site. Therefore, a model of DFA I was placed in the active site of αFFase1 E. In addpNP-α-D-Araf and inulobiose were measured  simulations starting from the β-D-Fruf complex structure and modeled complex structures with inulobiose and DFA I were performed for 1 μs. The whole hexamer of chains A to F was used for the simulation. The ligand in each active site was monitored. β-D-Fruf monosaccharides in the active sites of chains A, B, D, E, and F moved back and forth between −1 and +1 subsites, in which approximate distances between the anomeric C2 atom and the nucleophile were 3.5 and 5.0 Å, respectively (A). A β-D-Fruf molecule in chain C slipped out from the active site during 400 to 700 ns through the channel to the internal cavity (B). Thus, the average RMSD of the ligand in chain C during the entire MD run was significantly higher than that of the other chains (B). Similarly, DFA I in chains C, E, and A moved after 320, 550, and 850 ns, respectively, but those in the other chains remained at the canonical position (C). The snapshots of inulobiose in chain A and DFA I in chain D are shown in C and D, and their movies are shown in Figure 6ectively A. A β-D-l cavity B. Thus, r chains . Inulobir chains B. Similaposition C. The snpβ2,1Fru (C). Glu270 is located at an appropriate position for proton donation  to the O2 hydroxy group of α-Fruf at the −1 subsite, and Glu291 is placed at a position working as a nucleophile to the anomeric carbon (C2). After this step, the O2 hydroxy group is released from the substrate as a water molecule. (iii) Rotations of the glycosidic bond and the C1–C2 bond of the +1-subsite sugar are required for the intramolecular transfer reaction to occur. (iv) When the C1 hydroxy group of fructose at the +1 subsite is appropriately positioned for proton acceptance  by Glu270, deglycosylation of Glu291 is facilitated. (v) After the reaction at the active site, DFA I is released through the channel to the inner cavity of the hexamer of αFFase1 (A and B). The opposite reaction from DFA I (v) to inulobiose (i) is expected to be similar to the standard retaining reaction mechanism of GHs (https://www.cazypedia.org/index.php/Glycoside_hydrolases) . For GHs, Ca2+ often contribute to protein stabilization, as exemplified by GH13 α-amylases  Thepβ2,1Fru C. Glu270 αFFase1 , A and Brolases) . The foreraction D. For GHamylases . Severalbinanase , GH62 α-nosidase , GH47 annosidase , 41, GH9nosidase , and GH1f bond of DFA III, the reaction product is inulobiose with β-Fruf at the reducing end. The GH91 family of enzymes act on the β-Fruf linkages with the lytic transglycosidase-like mechanism  is available on the PDB from a structural genomics project, no functional information about DUF2961 is available. Importantly, αFFase1 does not show significant amino acid sequence similarity to any known enzymes, not just GHs. According to the results of this study, the Carbohydrate-Active enZymes team will create a new GH family, GH172 .αFFase1 belongs to the DUF2961 (PF11175) family in the Pfam database . AlthougD-fructofuranosidase and α-D-arabinofuranosidase activities. Arabinofuranosyl units in plant glycans are exclusively L-isomers, and α-D-arabinofuranosides are rarely found. For example, the cell wall polysaccharides of mycobacteria  or difructose compounds  present in the human diet. The main products of the caramelized D-fructose promoted by acid ion-exchange resins are DHL II and DFA I  did not use DFA III in vitro . BBDE_2040 (αFFase1) and BBDE_2039 (GH32) are highly likely intracellular enzymes because they do not have an N-terminal secretion signal sequence. The neighboring ABC transporter genes (BBDE_2041-2043) (A) seem to be responsible for importing substrates for αFFase1 into the B. dentium cell. BBDE_2039 or other GH32 β-D-fructofuranosidases in the genome  may cleave inulobiose into two D-fructose molecules.αFFase1 exhibited both α-bacteria  and corybacteria , pilins ruginosa , and liptophilia  were repd caries  and fromd caries , we specnd DFA I . B. dentt inulin , and fivin vitro . However41-2043) A seem to−128 (A), whose oligomerization architecture is clearly different from that of αFFase1 (A). Putative −1 subsite residues in BACUNI_00161, including the two catalytic glutamate residues, are conserved with those of αFFase1 (A and B). However, Cys304 appears to clash with the O2 atom of Fruf in the −1 subsite, and residues in the putative +1 subsite are not conserved, suggesting that the Bacteroides protein is not a DFA I synthase/hydrolase.Phylogenetic analysis of αFFase1 homologs was performed. Manual selection of homologs in currently available microbial genomes, in which the catalytic pocket residues are conserved, produced 120 sequences with sequence identity >47.45%, sequence coverage >79%, and e-value < 5.0 × 10−128  and S16.−128 A, whose  αFFase1 A. Putati αFFase1 , A and BA. fumigatus Af293 (XP_748288.1), which shares 48.6% sequence identity with BBDE_2040, is included in this fungal clade, suggesting that it corresponds to the A. fumigatus difructose-anhydride synthase studied in the 1980s . Moreover, the gene pairs BBDE_2040 and BBDE_2039 are present in the genomes of 6 of 10 strains of B. breve isolated from the feces of infants, adults, and elderly individuals . Th. ThA. fue.g., cyclodextrin, cyclic α-maltosyl-(1→6)-maltose, and cycloalternan) is often part of a monopolizing metabolic pathway that is advantageous in the competition for starch acquisition by transiently changing the polysaccharide into special cyclic forms  (D-fructofuranosidase (BN159_0194) are present in the genome, suggesting that S. davawensis uses inulin via a three-stage metabolic pathway via DFA I. However, in the B. dentium JCM 1195 genome, there are no putative GH91 inulin fructotransferase genes. The metabolic system for α-D-fructofuranoside-containing sugars in oral and intestinal bacteria may have emerged after humans began using fire to cook food. The metabolic system may be relevant to the opportunistic pathogenicity of B. dentium in the development of dental caries  . The putl caries . We willD-Fruf-(2→6)-D-Glc were kindly provided by Ohtaka Enzyme Co Ltd  and HPLC on a COSMOSIL PBr column . D-Fruf-α-Me and D-Araf-α-Me were synthesized by methanolysis, as described by Bethell and Ferrier  protected D-arabinofuranose in the furanoside form. This was followed by a one-pot treatment with acetic anhydride at room temperature overnight to obtain the corresponding 1,2,3-tri-O-acetyl-5-O-TBDPS protected D-arabinofuranose. After the introduction of thioglycoside at the anomeric position with 4-methylbenzenethiol in the presence of boron trifluoride etherate in dichloromethane at 0 °C, the TBDPS group was converted to acetate with tetra-n-butylammonium fluoride in tetrahydrofuran followed by acetic anhydride in pyridine (para-nitrophenol in NIS–silver(I) triflate in dichloromethane with MS 3A at 0 °C for 1 h afforded α-arabinofuranoside stereoselectively through neighboring group participation. Finally, conventional deprotection with NaOMe in methanol (MeOH) at 0 °C for 15 min gave pNP-α-D-Araf. The structure of the product was verified by 1H NMR in D2O as the α-isomer   .pNP-α-D-Fruf was synthesized using the method for the synthesis of pNP-α-D-Araf. The furanoside formation of D-fructose with tert-butyl(chloro)diphenylsilane in pyridine followed by a one-pot treatment with acetic anhydride afforded Fruf protected by 2,3,4-tri-O-acetyl-1,6-di-O-TBDPS. After the introduction of the thioglycoside two-step conversion of 1,6-di-O-TBDPS to the corresponding acetate, selective glycosylation with para-nitrophenol followed by conventional deprotection afforded α-D-Fruf. Finally, conventional deprotection with NaOMe in MeOH at 0 °C for 15 min gave pNP-α-D-Fruf after separation by preparative TLC (eluent:CHCl3–MeOH = 10:1) on silica gel. The structure of the product was verified as the α-isomer from the peak at 113.1 ppm for C2 in the 13C NMR spectra and the peak at 4.21 ppm  for C3-H in the 1H NMR spectra because C2 of α-Fruf generally appears in the lower field, and the coupling constant 3JH3–H4 of α-one is generally a smaller value than that of β-one, respectively . The primer sets indicated in E. coli BL21 (DE3) (Agilent Technologies) was transformed with the αFFase1 expression vector and cultured in the LB medium containing 100 μg/ml ampicillin at 37 °C until the absorbance at 600 nm reached 0.4 to 0.6. The gene expression was induced by the addition of IPTG at a final concentration of 0.1 mM, and the culture proceeded for another 20 h at 25 °C. The bacteria were recovered by centrifugation and suspended in a lysis buffer composed of 50 mM Tris HCl (pH 7.0) and 300 mM NaCl. The suspension was sonicated using a model 250D sonifier (Branson Ultrasonics Corporation). The lysate was centrifuged, and the supernatant was applied to a column filled with cOmplete His-tag Purification Resin (Roche Diagnostics GmbH). The column had been preequilibrated with the lysis buffer. The column was washed with the lysis buffer supplemented with 5 mM imidazole, and the protein was eluted with the lysis buffer supplemented with 500 mM imidazole. The eluate was collected and concentrated using Amicon Ultracel-10K centrifugal filters (Millipore). The sample was loaded onto a Hiload 16/60 Superdex 200 pg column (Cytiva), preequilibrated with 25 mM Tris HCl (pH 7.0) and 300 mM NaCl. Approximately 50 to 70 mg of the target protein per liter culture with a purity of >99% was typically obtained after the sequential column-purification steps. Fractions corresponding to the target protein were collected and concentrated, and the buffer was exchanged with 5 mM sodium acetate (pH 6.0) using Amicon Ultracel-10K centrifugal filters. The purified protein was stored at 4 °C at a concentration higher than 10 mg/ml until further use. Protein sample purity was verified by SDS-PAGE during purification and conservation and analyzed by GelAnalyzer v.19.1. The protein concentration was determined by the bicinchoninic acid assay using bovine serum albumin (TaKaRa Bio) as the reference standard protein. For crystallization sample, which required a higher concentration, the protein concentration was determined by measuring the absorbance at 280 nm using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) using the molar extinction coefficient of 107,175 M−1 cm−1 that was calculated from the amino acid sequence.The S-v plot was analyzed by linear curve regression using SigmaPlot 12.0.For every type of the substrate, enzyme activity was investigated by TLC and HPAEC-PAD. For TLC, a developing solvent of 1-butanol:2-propanol:water (10:5:4) was used, and orcinol sulfate was used as a visualizing solution. HPAEC-PAD was performed using the CarboPac PA1 column (Thermo Fisher Scientific). Elution occurred at a flow rate of 1.0 ml/min using the following gradient: 0 to 5 min, 100% eluent A (0.1 M NaOH); 5 to 30 min, 0 to 100% eluent B (0.5 M sodium acetate and 0.1 M NaOH); and 30 to 35 min, 100% eluent B. Carbohydrate concentrations were adjusted for the peak areas of standard samples. Calibration curves for DFA I and inulobiose up to 10 μM  and 2 μMInulobiose was also used to determine the effect of metal ions on the ligand and enzyme activity. αFFase1 was incubated for 24 h at 4 °C with the solution described above that was depleted of inulobiose and supplemented with 1 mM of divalent ions. Inulobiose was added, and the reaction occurred at 37 °C.pNP-α-D-Araf, the activity was monitored by stopping the reaction with 4 volumes of 1 M sodium carbonate. The amount of released pNP was spectrophotometrically determined by measuring the absorbance at 400 nm. The reaction solution was composed of 0.1 μg/ml (1.89 nM) αFFase1, 100 mM sodium acetate (pH 6.0), and different concentrations of pNP-α-D-Araf at 37 °C. The S-v plot was fit to the Michaelis–Menten equation using the enzyme kinetics tool from SigmaPlot 12.0. pNP-α-D-Araf was also used to determine the optimal pH and temperature of αFFase1.For D-fructose (60 g) at 130 °C for 120 min. For the mixture of fructose and glucose, 30 g of D-fructose and 30 g of D-glucose were used. The obtained solution was diluted in distilled water to a final concentration of 300 g/l. The remaining D-fructose was consumed by a baker's yeast fermentation process. Frupβ2,1Fru and DHL II were separated from fructose caramel by gel-filtration chromatography on a Bio-Gel P-2 column  equilibrated with 1% sodium acetate. Subsequently, the oligosaccharide was purified using a combination of an Autoprep FiberAC activated carbon column (Showa Denko) and a COSMOSIL PBr column  preequilibrated with 20 mM sodium phosphate (pH 2.5). Finally, Frupβ2,1Fru and DHL II were purified using a COSMOSIL PBr column with water as the mobile phase.Caramelized sugar was prepared, as previously described  by heati2O were analyzed by NMR (3 for further verification (m/z = 346.5 Da) of the disaccharide (m/z = 364.5 Da). MALDI-TOF mass analysis of both nonacetylated and acetylated fractions was also performed to estimate the number of hydroxy groups in the sample at peak 1  and peak 3 . The findings suggested that the anhydride was afforded from the disaccharide as the product (The collected fractions (peak 1 purified from caramelized sugar and peak 3 after treatment with αFFase1) were concentrated by freeze-drying, and the samples in Dfication . MALDI-T product .pNP-α-D-Araf in D2O. A portion of the substrate (600 μl of a 10 mM solution in D2O) was mixed with 1 μl of the enzyme solution (30 mg/ml or 566 μΜ in water). The 1H NMR spectra of the reaction mixtures were recorded at 37 °C using an ECX400 spectrometer (JEOL) operating at 400 MHz (A). The 1H NMR spectra of the authentic samples, such as the initial pNP-α-D-Araf and D-arabinose, which reached equilibrium, were obtained without enzyme addition. Using the spectra of the hydrolysis after 1 min, the 1H data of α-D-Araf in the mixture were corrected and assigned using the relationship of the 3JH–H coupling constants shown in f in the reaction mixture was clearly observed in the anomeric region as the initial major furanoside (B).The hydrolysis reaction was performed with 10 mM  400 MHz A. The 1Hranoside B.B) and NMR (A). A portion of inulobiose or enzymatically obtained DFA I  was mixed with 50 μl of the enzyme solution in 50 mM sodium phosphate buffer (pH 6.0), with a total volume of 600 μl. The molar ratio of DFA I to inulobiose was monitored by the time course of the reaction of αFFase1 on HPAEC-PAD. The 1H NMR spectra of the reaction mixtures after 0 min  and 24 h (after equilibrium) were recorded at 37 °C in the same manner as Figure 2A. The spectra are presented in Figure S10A. The data of the product (DFA I) shown in Figure S10B unambiguously indicated by 1JH3–H4 (2.4 Hz) and 13C chemical shifts of C3 at 82.1 ppm that the →3)-α-Fruf structure was formed from inulobiose.Monitoring of the equilibrium between inulobiose and DFA I was performed using HPAEC-PAD  and 4B a4 and NMR A. A portD-arabinose or 20 mM D-fructose. The crystals were cryoprotected in a reservoir solution supplemented with 5%–10% 2-methyl-2,4-pentanediol and flash-cooled by dipping in liquid nitrogen.Crystallization was performed by the sitting drop vapor diffusion method at 20 °C by mixing a protein solution  with a reservoir solution containing 20% (w/v) PEG 3350 and 0.2 M disodium tartrate at a 1:1 volume ratio. Cocrystals were prepared using a protein solution containing 80 mM Diffraction data were collected on beamlines at the Photon Factory of the High Energy Accelerator Research Organization (KEK) and Swiss Light Source (Paul Scherrer Institute). The diffraction datasets were processed using XDS  and Aimlhttps://molview.org) was placed in the active site of αFFase1 by superimposing the five-sugar ring atoms of one Fruf onto the β-Fruf molecule in the complex structure. Furanose conformations of the model were 4T5 (p = 30.6°) and E3 (p = 342.2°) for the sugars in −1 and +1 subsites, respectively. The model was energy-minimized using the sculpting wizard of PyMOL.A model of DFA I retrieved from MolView (E. coli JM109 (Agilent Technologies) for homologous recombination and amplification. The plasmid was then extracted, and the sequence was checked before being transformed into E. coli BL21 Star (DE3) (Invitrogen). Production and purification of the mutant proteins were performed in the same manner as the WT protein. Enzymatic activity was studied as described above, except that the substrate concentration was fixed at 2.5 mM for pNP-α-D-Araf and 10 mM for inulobiose.Site-directed mutagenesis was performed using the complementary primer sets listed in d-Fruf complex, αFFase1–inulobiose complex, and αFFase1–DFA I complex. The initial structures of the MD simulations were prepared in the same manner, except that the crystal structure was used for the MD simulation of the αFFase1–β-d-Fruf complex, and the modeled structures were used for the MD simulations of the other two complexes. In each initial structure, the entire hexameric structure was used and the ligand was bound to all the protomers. The N and C termini of each protomer were capped with acetyl and N-methyl groups, respectively. The protonation states were determined based on the hydrogen bond network. His16, His207, and His262 were protonated at the Nδ1 atoms, and His 344 was protonated at both the Nδ1 and Nε2 atoms. The other histidine residues were protonated at the Nε2 atoms. After these modifications, the structure was immersed in a cubic box of water, ensuring a minimum distance of 10 Å between any box face and any protein atom. Potassium ions were added, so that the net charge of the system was zero. The dimensions of each system were 151 × 151 × 151 Å and comprised six identical protein chains, six identical ligands, 102 potassium ions, and approximately 88,000 water molecules. The Amber ff14SB force field parameters .Atomic coordinates and structure factors  have been deposited in the Protein Data Bank (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions. Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases have attracted attention for their application as biocatalysts due to their flexibility and high efficiency. Here, the authors show the biochemical and structural characterizations of the versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. In animals, Fe/αKG oxygenases play important roles in physiological processes via hydroxylation or N-demethylation reactions, with substrates including proteins, nucleic acids, and lipids1. In plants and microorganisms, in addition to the typical hydroxylation reactions, Fe/αKG oxygenases catalyze a wide range of chemical transformations in the biosynthesis of natural products27, including complex skeletal rearrangement12, ring-expansion15, and C–C bond formation23. Given their intriguing chemical reactions, detailed biochemical, structural, and calculation studies of Fe/αKG oxygenases have been conducted over the past few decades32.Oxygenases are widely distributed in nature and catalyze the incorporation of oxygen into various natural products. One of the largest oxygenase groups is the non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenase superfamily, which is involved in both primary and secondary metabolism35. Through the oxidative decarboxylation of αKG, a reactive Fe(IV)-oxo intermediate is generated to activate a selective aliphatic C–H bond, which is normally difficult to cleave due to high bond-dissociation energy, and then oxidative products are generated following radical recombination. Unlike other oxidases, including P450 monooxygenases, Fe/αKG oxygenases do not require any reductase partners or expensive cofactors such as NAD(P)H and FAD/FMN36. Furthermore, these enzymes exhibit high catalytic efficiencies, and typically have large turnover numbers38. These features of Fe/αKG oxygenases, along with their catalytic versatility, have attracted considerable attention as a source of biocatalysts39.Fe/αKG oxygenases utilize αKG as a co-substrate and Fe(II) as a cofactor40. It catalyzes two oxidation steps to form emervaridone C (3) from andiconin D (1) natural substrates, along with our structure-based mutagenesis studies, have revealed the molecular basis for the substrate recognition and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity.In this study, we investigated SptF further by in vitro enzyme reactions, crystallization, and structure-based mutagenesis. To our surprise, SptF is an unusually promiscuous and catalytically versatile enzyme that performs up to four rounds of oxidative reactions with its natural substrate 3) was previously reported as the final product of SptF from andiconin D (1)40. However, our careful reexamination of the enzyme reaction revealed that SptF actually produced two more products 4 and 5, in addition to 2, 3, and previously reported emeridone F40  from andilesin C (6), in the closely related anditomin pathway , +2 of 3 (m/z 428 [M + NH4]+) was observed (m/z 426 (+0): 428 (+2) = 2.2%: 97.8%). On the other hand, in the experiment with H218O , small amount of 18O was incorporated into 3 (m/z 426 (+0): 428 (+2) = 95.6%: 4.4%). Further, double labeling of H218O and 18O2 revealed that the enzyme generated +2 of 3 (m/z 428) as a major product together with +0 (m/z 426) and +4 (m/z 430) of 3 as minor products (m/z 426 (+0): 428 (+2): 430 (+4) = 1.3%: 92.0%: 6.8%). These results indicated that an epoxide group introduced by SptF is mostly from O2, while ~5% of water-derived oxygen atom are also incorporated into 3. This would be caused by solvent exchange of ferryl and/or ferric species with water during the catalytic cycle41 and/or nonenzymatic reaction of 3 with water molecule. Indeed, the incubation of 3 with 78% H218O without SptF revealed the incorporation of an oxygen atom from water (m/z 426 (+0): 428 (+2) = 94.6%: 5.4%), indicating that an oxygen atom of 3 can be nonenzymatically exchanged with water. A possible explanation is addition of water to a carbonyl group to yield a geminal-diol. Its collapse back to the carbonyl could proceed with elimination of 16O water, explaining the 18O/16O exchange. This reaction may happen at C4’ carbonyl of 3. This carbonyl carbon becomes less reactive in 4, because the C9 hydroxyl group blocks it, and also less reactive in 5, where it is turned into an ester carbonyl. In fact, the observed 18O/16O exchange was not so significant in 4 and 5 as it was in 3.To investigate the origin of oxygen atoms incorporated by SptF, we performed labeling experiments using m/z 447) were observed for 4 (m/z 443 (+2): 445 (+4): 447 (+6) = 1.1%: 8.6%: 90.3%) and 5 (m/z 443 (+2): 445 (+4): 447 (+6) = 0.9%: 3.4%: 95.7%) when we incubated 1 and SptF with 98% 18O2. Furthermore, the double labeling experiments with 78% H218O and 98% 18O2 showed similar labeling pattern to 98% 18O2-labeling experiment (for 4 (m/z 443 (+2): 445 (+4): 447 (+6): 449 (+8) = 0.9%: 4.8%: 92.1%: 2.2%) and for 5 (m/z 443 (+2): 445 (+4): 447 (+6): 449 (+8) = 0.8%: 3.1%: 95.1%: 1.0%)). These results clearly indicated that all oxygen atoms incorporated into 4 and 5 are mostly derived from O2. In contrast, the very small +8 peaks of 4 and 5 were observed in H218O-labeling and double labeling experiments.On the other hand, major +6 peaks (10), preandiloid C (12), terretonin J (13), terretonin C (15), terretonin A (17), and terretonin (19) , testosterone (24), and progesterone (28), as substrates. To our surprise, SptF accepted 21, 24, 28 of them with high conversion rates of 34%-53% over 24 h /6, and SptF S114A-Fe/NOG/15 , 9 residues , 16 residues (Asn54-Lys69), and 19 residues (Trp53-Lys71) were constructed  , Merck KGaA Ltd. , and Hampton Research , unless noted otherwise. PCR was performed using a TaKaRa PCR Thermal Cycler Dice® Gradient (Takara Bio Inc.). The NMR spectra of compounds were recorded on Bruker AVANCE III HD 800 MHz, AVANCE III HD 900 MHz , ECX-500 MHz, and EXZ-500 MHz  spectrometers.Oligonucleotide primers, listed in Supplementary Table 50, were prepared as follows. The primer pair SptF-N3-F/SptF-N3-R was used for amplification of the SptF DNA fragment from the previously reported pET-28a(+)-sptF40 plasmid (see Supplementary Table NdeI/HindIII-digested pET-22b (+) vector, using an In-Fusion® HD Cloning Kit (TaKaRa Clontech). The identities of the resulting vectors were confirmed by DNA sequencing. Escherichia coli DH5α was used for plasmid construction, and E. coli RosettaTM2(DE3)pLysS (Novagen) was used for the preparation of recombinant proteins.To obtain stable and pure proteins for in vitro enzyme reactions and crystallization experiments, expression plasmids for SptF lacking three residues, predicted to be flexible by XtalPred server40 plasmids. SptF PCR products were purified and ligated into the NdeI/HindIII-digested pET-22b (+) vector, using an In-Fusion® HD Cloning Kit (TaKaRa Clontech). The identities of the resulting plasmids were confirmed by DNA sequencing.The primers used for the construction of plasmids for site-directed mutagenesis studies are listed in Supplementary Table E. coli RosettaTM2(DE3)pLysS (Novagen). The resulting strains were cultured in LB medium supplemented with 34 mg/L chloramphenicol and 50 mg/L ampicillin sodium, with shaking at 37 °C. When the OD600 reached 0.6, the cell cultures were cooled down on ice for 30 min, and then IPTG (0.2 mM) was added to induce the target protein expression at 16 °C. After 18 h of post-induction incubation, cells were harvested by centrifugation at 3300 × g for 15 min and suspended in lysis buffer, containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 5% glycerol. The cell suspension was sonicated for 4 min on ice. After removal of the cell debris by centrifugation at 20,400 × g for 30 min, the supernatant was mixed with 1 mL Ni-NTA agarose resin and loaded onto a gravity flow column. Unbound proteins were removed with 50 mL lysis buffer containing 25 mM imidazole, and then the His-tagged protein was eluted with lysis buffer containing 300 mM imidazole. For the in vitro assay, the protein eluate was concentrated to 10 mg/mL after the removal of imidazole, using a 30 kDa Amicon® Ultra-15 filtration unit (Millipore). For crystallization, the eluate from the Ni-NTA agarose was applied to a HiLoad 16/60 Superdex 200 prepacked gel filtration column , and eluted with a solution containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM dithiothreitol. The resulting eluate was concentrated to 10 mg/mL, using an Amicon Ultra-4 (MWCO: 30 kDa) filter at 4 °C. The purity of the purified proteins was monitored by SDS-PAGE . and the proteins were prepared as described above. All reactions in this study were performed following the previous studies unless noted otherwise40. Reactions were performed on a 50 μL scale with 50 mM PIPES (pH 7.5), 0.2 mM FeSO4, 5 mM α-ketoglutarate, 4 mM ascorbate, 100 μM of substrate, and 15 μM of wild-type or mutant SptF. The reaction mixture without protein was used as the negative control. Enzyme reactions, except for steady-state enzyme kinetics, were incubated at 30 °C for 1 h or overnight and quenched by adding 50 µL methanol. After centrifugation and filtration, the reaction mixtures were analyzed by LC-MS. For comparisons of the substrate consumption between truncation variants and wild-type, the reactions were performed on a 50 μL scale with 50 mM PIPES (pH 7.5), 0.2 mM FeSO4, 5 mM α-ketoglutarate, 4 mM ascorbate, 100 μM of substrate, and 40 μM of enzyme.Substrates used for in vitro enzyme reactions, including 52. Reactions were performed under anaerobic conditions in 500 μL tubes, in which 25 μL solutions, containing 5 μM SptF enzyme, 200 μM 1, 5 mM αKG, 4 mM ascorbate, 200 μM FeSO4, and 40 mM PIPES buffer (pH 7.5). Afterward, 18O2 gas (98%) or air was injected into the vial. For the H218O labeling experiment, 20 mL of H218O (97%) was added for the preparation of 25 μL reaction solutions . The enzyme reactions were performed at 20 °C for 10 min and quenched by adding equal volume of methanol. The prepared sample was stored at −80 °C until LC-MS analysis to decrease the exchange rate with solvents. The products were analyzed with the same method as that for the standard reaction products.The labeling experiments were performed using previously reported methods2O supplemented with 0.1% v/v formic acid as solvent A and acetonitrile as solvent B. The gradient for the separation of compounds in reaction mixtures with 1, 10, 12, 17, or 19 was 20% to 45% B over 20 min, then increase to 100% B in 1 min, and hold for 4 min at 40 °C with a flow rate of 0.2 mL/min. Reaction mixtures with 6 are shown here: 20–60% B over 17 min, then increase to 100% B in 0.1 min, and hold for 2.9 min at 40 °C with a flow rate of 0.15 mL/min. Reaction mixtures with 13 or 15 are shown here: 20–100% B over 20 min, and hold for 5 min at 40 °C with a flow rate of 0.15 mL/min. Reaction mixtures with 21, 24, or 28 are as follows: 10–100% B over 20 min, and hold for 5 min at 35 °C with a flow rate of 0.15 mL/min.All samples from in vitro assays were analyzed by a Bruker Compact QqTOF mass spectrometer (Bruker) coupled with an LC-20AD ultra-high-performance liquid chromatography (UHPLC) system (Shimadzu). MS data were recorded in the positive electrospray ionization mode. Compounds were separated on a COSMOSIL 5C18-MS-II Packed Column . All reaction mixtures were separated using H4 and 5, and has better diffraction as compared with wild-type SptF, we employed it for the soaking experiments of 15. The complex structures were prepared by incubating SptF, N65T, or S114A crystals at 10 °C for 12 h with 5 mM of compound, 5 mM of αKG/NOG, and 100 µM of Fe in the crystallization drop. When αKG was used, the soaking experiment was performed in an anaerobic chamber.Crystals of SptF lacking three residues, as well as the N65T and S114A mutants, were obtained after 1 day at 10 °C by using the sitting-drop vapor-diffusion method with the following reservoir solutions: SptF-apo: 0.27 M magnesium chloride, 21% w/v PEG 3350, 1.5% trimethylamine N-oxide dihydrate. SptF N65T: 0.2 M magnesium formate dehydrate, 20% w/v PEG 3350. SptF S114A: 0.27 M magnesium chloride, 20% w/v PEG 3350, 0.2 M sodium chloride, 1.5% trimethylamine N-oxide dihydrate. The protein concentrations used for the crystallizations of SptF, N65T, and S114A were 7.5 mg/L, 10 mg/L, and 7.5 mg/L, respectively. Since S114A can produce 53 and Aimless in ccp454. The initial phase of the SptF structure was determined by molecular replacement, using AndA (PDB ID: 5ZM4) as the search model. Molecular replacement was performed with Phaser in PHENIX56. The initial phase was further calculated with AutoBuild in PHENIX57. The SptF structures were modified manually with Coot58 and refined with PHENIX.refine59. The cif parameters of the ligands for the energy minimization calculations were obtained by using the PRODRG server60. The ligands were added in the unassigned density at active site with several conformations and compared their 2mFo-DFc and mFo-DFc maps and Real-space fit  values calculated by PHENIX.refine and PDB validation server, respectively  glycerol), and then flash cooled at −173 °C in a nitrogen-gas stream. The X-ray diffraction datasets were collected at BL-1A , using a beam wavelength of 1.1 Å;. The diffraction datasets for SptF were processed and scaled using the XDS program package1 and αKG, 98.5% favored, 1.5% allowed for SptF complexed with 6 and NOG, 97.7% favored, 2.3% allowed for SptF N65T complexed with 1, 98.3% favored, 1.7% allowed for SptF S114A complexed with 15 and NOG, and 98.5% favored, 1.5% allowed for SptF Δ9 complexed with NOG. All crystallographic figures were prepared with PyMOL .The Ramachandran statistics are as follows: 98.3% favored, 1.7% allowed for SptF-apo, 97.7% favored, 2.3% allowed for SptF complexed with 4, and 1 mM αKG, at 30 °C, and quenched by the addition of 50 μl methanol. Compounds 1 (0.5–25.0 µM), 6 (0.5–20.0 µM), and 15 (10.0–300.0 µM) were used to incubate with 0.025, 0.01, and 5.0 µM SptF for 3 min, 5 min, and 2 h to determine the kinetic parameters, respectively. Similarly, compounds 1 (1.0–100.0 µM) and 15 (10.0–200.0 µM) were used to incubate with 0.5 and 5.0 µM N65A for 5 min and 2 h to determine the kinetic parameters, respectively. For F133A, compounds 1 (20.0–300.0 µM) and 6 (10.0–200.0 µM) were used to incubate with 5.0 and 2.5 µM enzyme for 30 min, respectively. For I63A or loop-truncation Δ9 mutants, 5.0 µM enzyme was used to incubate with 15 (10.0–300.0 µM) for 2 h. Detailed concentration of substrates used to determine the kinetics of each enzyme were shown in the plot of Supplementary Table 1 and 6, kinetic parameters were determined based on consumption of substrates, while for 15, formation of product was used instead. Each reaction was performed in triplicate. GraphPad Prism  was used for statistics data analysis , 4 mM ascorbate, 200 μM FeSO1, 6, 10, 13, and 15 for structure determination, in vitro enzymatic reactions were performed as described above in a total volume of 100 mL, divided into 0.5 mL portions in 2 mL tubes. After an overnight incubation at 30 °C, the reactions were quenched and extracted twice with an equal volume of ethyl acetate. After the ethyl acetate removal, 1 mL methanol was added to dissolve the products for separation by semi-preparative HPLC, which was performed on a Shimadzu LC20-AD HPLC system.To isolate the products from 4, 5, and 33 were purified from large scale enzymatic reactions as mentioned above, using wild-type SptF and the F133A mutant with 1. Compounds 8 and 9 were prepared using wild-type SptF from 6. These products were purified on an XSelect HSS T3 OBD Prep column  using 50% methanol supplemented with 0.1% v/v formic acid at 40 °C. The injection volume was 0.1 mL and the flow rate was 2.5 mL/min. Finally, 0.3–0.4 mg of compounds 4, 5, 8, 9, and 33 were obtained.Compounds 32 was purified from large scale enzymatic reactions using I231A, on an XSelect HSS T3 OBD Prep column  with H2O supplemented with 0.1% v/v formic acid as solvent A and methanol as solvent B, at 40 °C. The gradient for separation was 46% B for 40 min, and 56% B for 60 min. The injection volume was 0.1 mL and the flow rate was 2.5 mL/min. The purification yielded 0.3 mg of compound 32.Compound 11 was purified with an XSelect HSS T3 OBD Prep column , using 45% acetonitrile supplemented with 0.1% v/v formic acid, at 40 °C. The injection volume was 0.15 mL and the flow rate was 3.0 mL/min, and 0.9 mg of compound 11 was obtained.Compound 11 was purified with an XSelect HSS T3 OBD Prep column , using 45% acetonitrile supplemented with 0.1% v/v formic acid, at 40 °C. The injection volume was 0.15 mL and the flow rate was 3.0 mL/min, and 0.9 mg of compound 11 was obtained.Compound 16 was purified with a COSMOSIL πNAP Packed column , using 50% acetonitrile supplemented with 0.1% v/v formic acid, at 40 °C. The injection volume was 0.15 mL and the flow rate was 3.0 mL/min, and 0.9 mg compound 16 was obtained.Compound 4, 4 mM α-ketoglutarate, 2 mM ascorbate, 1.6 mM of substrate, 10% glycerol, and 50 μM of SptF, at 4 °C for 56 h. Products were separated by normal-phase silica gel chromatography. The elution conditions were as follows: CHCl3: MeOH = 100: 0.5 for 540 mL; then CHCl3: MeOH = 100: 1 for 90 mL, and CHCl3: MeOH = 100: 1.5 for 90 mL. Finally, 2.0 mg and 1.3 mg of compounds 22 and 23 were obtained from 21, respectively. From 24, 2.2 mg and 1.3 mg of 25 and 26/27 were obtained, respectively.Reactions were performed in a total volume of 100 mL, with 100 mM HEPES (pH 7.5), 0.2 mM FeSO2)3(tpt)2(cyclohexane)x] -1,3,5-triazine)61 was used as the crystalline sponge in this study. The crystalline sponge  was placed in a vial and immersed in a small amount of cyclohexane (50 μL). A 1,2-dichloroethane solution (5 μL) of the compound  was added to the vial. The vials were equipped with a syringe needle for slow solvent evaporation. Guest soaking was carried out at 50 °C for 1 d. After confirming solvent evaporation, the X-ray diffraction datasets were collected at BL02B1 , using a beam wavelength of 0.4115 Å. The sample crystal was cooled to 100 K, using a cold nitrogen stream. The collected data were integrated, corrected, and scaled using the program CrysAlisPro, ver. 1.171.38.46 . All crystal structures were solved using the program SHELXT ver. 2014/562 and refined with the program SHELXL ver. 2018/163. The crystal structure of the 25/crystalline-sponge complex was solved and three independent molecules of 25 per asymmetric unit were clearly observed. All nonhydrogen atoms were refined anisotropically. All hydrogen atoms were generated using the proper HFIX command and refined isotropically using the riding model. All nonhydrogen atoms in the framework were refined without restraints and constraints. Guest molecules were refined with restraints on the atomic displacement parameters (SIMU and RIGU). Three independent guest molecules were characterized as geometrically equal molecules by restraint (SAME). The populations of the three independent guest molecules per asymmetric unit were estimated to be 69%, 56%, and 31%, according to the structure refinement. The solvent cyclohexane molecules suggested in the electron density map were refined using some restraints , because the corresponding electron densities were obscured due to their thermal motion and disorder. The absolute configuration of 25 was thus established as 8 S, 9 R, 10 S, 13 S, 14 S, 17 S, based on the Flack [0.122 (10)] parameters. Crystallographic data are as follows. Refined formula: C118.33H125.04Cl12N24O4.68Zn6, formula weight (Mr): 2775.94, crystal system: monoclinic, space group: C2, Z = 4, Rint = 0.0634. Lattice parameters, R-factor on F2 > 2σ(F2), and weighted R-factor are as follows: a = 32.8346(8) Å, b = 14.4445(2) Å, c = 30.7569(8) Å, β = 100.966(2)°, V = 14321.0(6) Å3, R = 0.0977, wR = 0.3103, and S = 1.197. The calculated density is 1.287. The linear absorption coefficient (μ) is 0.288. The Flack parameter, determined using 12,777 intensity quotients (Parsons’ method), is 0.122(10).A single crystal of [(ZnClFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The synthesis, single-crystal X-ray crystal structure, and magnetic properties are discussed.In this work, we present the complex salt  tetrabromido(ethanol/methanol-κO)nitrosylrhenate(II), [Ru(CN)(C5H5)2(C6H12N3P)4][ReBr4(NO)(CH4O)0.5(C2H6O)0.5] or [RuCp(PTA)2–μ-CN–1κC:2κ2N-RuCp(PTA)2][Re(NO)Br4(EtOH)0.5(MeOH)0.5]  was obtained and characterized by single-crystal X-ray diffraction, elemental analysis and infrared spectroscopy. The title salt was obtained by liquid–liquid diffusion of methanol/DMSO solutions of (NBu4)[Re(NO)Br4(EtOH)] and [(PTA)2CpRu–μ-CN–1κC:2κ2N-RuCp(PTA)2](CF3SO3). The RuII and ReII independent moieties correspond to a binuclear and mononuclear complex ion, respectively. A deep geometrical parameter analysis was performed, and no significant differences were found with earlier reports containing similar molecules. The magnetic properties were investigated in the temperature range 2.0–300 K, and the complex behaves as a quasi-magnetically isolated spin doublet with weak antiferromagnetic interactions.A new Ru Most of them fit the general formula [{RuCp(PTA)2–μ-CN–1κC:2κ2N-RuCp(PTA)2}-μ-mMX]n  2](CF3SO3) in the reaction with other transition-metal cation salts or complexes, in an easy, robust and reproducible method  or RAPTA complexes are known in inorganic medicinal chemistry for their potent antitumor activity et al., 2011et al., 2009et al., 20144)[ReII(NO)Br4(L)]  2][Re(NO)Br4(EtOH)0.5(MeOH)0.5]. The synthesis, single crystal X-ray crystal structure, and magnetic properties are discussed.On top of that, rhenium nitrosyl complexes applications are widely recognized: catalysis, production of organonitrogen compounds, pollutant control, nitric oxide release drugs, assembly of devices with novel optical and magnetic properties, among other uses 2][Re(NO)Br4(EtOH)0.5(MeOH)0.5] consists of discrete [RuCp(PTA)2–μ-CN–1κC:2κ2N-RuCp(PTA)2]+ cations and [Re(NO)Br4(EtOH)0.5(MeOH)0.5]− anions ns Fig. 1, which cII complex with two piano-stool fashion {RuCp(PTA)2} moieties that are linked by a –CN– bridging ligand. The {CpRu(PTA)2}+ moieties in each Ru2 unit exhibit a transoid arrangement related to the Ru—C≡N—Ru axis. The Ru1—C25 and Ru2—N13 distances are 2.008 (7) and 2.030 (8) Å, respectively. The Ru—CN—Ru arrangement is practically linear: <(Ru1—C25—N13) = 175.5 (7)° and <(C25—N13—Ru2) = 176.3 (7)°. The C≡N bond length of the cyano group is 1.14 (1) Å. The distances from the centroid of each Cp ligand to the respective ruthenium atom are 1.886 Å (Cp—Ru1) and 1.878 (Cp—Ru2). The Ru—PPTA distances are in the range 2.243 (2)–2.281 (2) Å, which is in agreement with those found in similar compounds.The cation is an homobinuclear RuII atom and displays a distorted octahedral geometry formed by four bromide ions in the equatorial plane, one nitrogen atom from the nitrosyl ligand, and one oxygen atom from an –OH group in apical positions. The –OH group comes from a methanol or an ethanol molecule, both with an s.o.f. of 0.5. The O1M and C1E atomic positions are the same for both the MeOH and the EtOH ligand. The Re1—O1m—C1e angle is 128.3 (6)°. The NO group is practically linear with an O101—N101—Re1 angle of 178.6 (10)°. The three atoms are also aligned with the O1M atom of the alcohol ligand, exhibiting a N101—Re1—O1M angle of 178.9 (3)°. The rhenium atom is shifted from the main plane of Br ligands towards the apical NO group by 0.157 Å.The complex anion is constituted by an ReP21/c space group. The cations interconnect adjacent anions via O—H⋯N hydrogen bonds and C—H⋯Br interactions, forming an infinite three-dimensional framework   Å, respectively. The angle defined by O1M—H1M⋯N8i is 165.5°.The complex crystallizes in the monoclinic k Table 1. The O—Hz) Fig. 2. The H1M2–μ-CN–1κC:2κ2N-RuCp(PTA)2]+ and bromides from [Re(NO)Br4(EtOH)0.5(MeOH)0.5]− units. The multiplicity and lack of defined directionality in the hydrogen-bond network are related to the fact that the major forces that stabilize the crystal are of electrostatic origin. The C—H⋯Br and the C⋯Br distances range from 2.53–3.12 Å and 3.208 (11)–3.944 (12) Å, respectively. The hydrogen-bond angle involving the C—H⋯Br atoms vary between 127 and 169°. These geometrical values are in concordance with weak hydrogen-bonding interactions Crystal Explorer17 .To further understand the intermolecular interactions between the ionic complexes within the crystal structure, a Hirshfeld surface  region of  reflects the significant amount of H⋯H contacts in the structure. Nevertheless, it is important to point out that the H⋯H contacts are usually difficult to localize in the Hirshfeld surface as they are spread all over the crystal packing. The Hirshfeld surface analysis for the cationic unit and its fingerprint also shows how H⋯N, N⋯H, H⋯Br, and H⋯H contacts surround the [RuCp(PTA)2–μ-CN–1κC:2κ2N-RuCp(PTA)2] unit. The relative contributions of the different intermolecular contacts to the Hirshfeld area for both ions are shown in Fig. 4The anion Hirshfeld surface shows how the most significant interaction is due to the O1m—H⋯N8 bond, which is illustrated by bright-red spots in Fig. 3et al., 20162CpRu-μ-CN-RuCp(PTA)2} moiety has been reported previously, once as an independent cationic unit in VOHCUS  version 5.42 in the last update of February 2021 .Regarding the anionic unit, examples of crystal structures containing tetrabromonitrosylrhenium(II) complexes are scarce. The CSD search yielded 19 hits. In all of them, the rhenium coordination sphere exhibits an octahedral geometry, with a practically lineal {Re—NO} unit and a π-acceptor ligand such as phosphine or aromatic amines, usually coordinating M—O—C angle lying in the range 123.333–130.865° . The same angle for metals coordinating an EtOH is in the range 124.464–132.412° (without considering possible outliers), in a total of 3503 reported structures. There are only five structures reported in the database containing ethanol coordinating to a rhenium atom, ABENRE ]− unit dates back to 1975  confirm that these H⋯Br contacts, considering the distance/angle criteria, can be identified as hydrogen bonds . Compounds containing a C—Br⋯H angle of less than 90° were discarded, as the hydrogen atom in the hydrogen bond must not point away from the acceptor atom (Aakeröy dc magnetic fields of 500 G (T < 20 K) and 5000 G (T ≥ 50 K). Experimental susceptibilities were carefully corrected for the diamagnetism of the holder (gelatine capsule) and constituent atoms by applying Pascal’s constants.Magnetic susceptibility measurements on polycrystalline samples were carried out with a Superconducting Quantum Interference Design (SQUID) magnetometer in the temperature range 2.0–300 K. In order to avoid saturation phenomena, we used external 2–μ-CN–1κC:2κ2N-RuCp(PTA)2][Re(NO)Br4(EtOH)0.5(MeOH)0.5] is shown in Fig. 5MT versus T plot where χM is the molar magnetic susceptibility per one ReII ion and T the absolute temperature. As expected, a straight line is observed for this compound  and a temperature independent paramagnetic contribution (TIP). The χMT value at room temperature is higher than that expected for an S = ½ with g = 2.0 (0.375 cm3 K mol−1) due to the temperature-independent paramagnetism (TIP). The slight decrease below 10 K must be attributed to very weak intermolecular antiferromagnetic (AF) interactions between the [Re(NO)Br4(EtOH)0.5(MeOH)0.5]− anions.The magnetic behaviour of  and [RuCp(PTA)2–μ-CN–1κC:2κ2N-RuCp(PTA)2](CF3SO3) were prepared as previously reported [Re(NO)Br4(EtOH)]  dissolved in 5 mL of a methanol–DMSO  mixture was layered in an test tube with a solution of [RuCp(PTA)2–μ-CN–1κC:2κ2N-RuCp(PTA)2](CF3SO3)  in 5 mL of the same methanol–DMSO mixture; ca 5 mL of the solvent mixture should be added between the two reactant layers to decrease diffusion time. Deep reddish-brown plate-like crystals, suitable for single crystal X-ray diffraction were obtained after one week. The product was filtered and washed by decantation with methanol. Yield: 24%. Analysis calculated for Ru2C36.5N14Re1O2Br4H63P4: C, 28.07; H, 4.07; N, 12.56; S. 0,00%. Found: C, 27.18; H, 4.39; N, 12.53; S. 0,00%. Selected IR absorption bands : 3413, 2922(w), 2114, 1759, 1280(m), 1242(s), 1097(m), 1016(s), 970(s), 948(s), 833(w), 744(w), 574(m), 480(m).A solution of (NBuUiso(H) = 1.2Ueq(C). Methanol/ethanol coordinating molecules were treated as positionally disordered utilizing the PART instruction with occupancy fixed to 0.5 applied to C1E, C1M, and C2E. C1M and C1E were constrained to occupy equivalent positions. Meanwhile, C2E was located in the Fourier difference map and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021006381/ex2046sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021006381/ex2046Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021006381/ex2046sup3.pngHistogram showing the number of Re-Br distances hexacoordinated nitrosylrhenium complexes found in the database survey (section 5). DOI: Click here for additional data file.10.1107/S2056989021006381/ex2046sup4.pngHistogram showing the number of Re-N(NO) distances in hexacoordinated nitrosylrhenium complexes found in the database survey (section 5). DOI: Click here for additional data file.10.1107/S2056989021006381/ex2046sup5.pngHistogram showing the number of N-O nitrosyl distances in hexacoordinated nitrosylrhenium complexes found in the database survey (section 5). DOI: Click here for additional data file.10.1107/S2056989021006381/ex2046sup6.pngHistogram showing the number of C...Br distances in hexacoordinated nitrosylrhenium complexes found in the database survey (section 5). DOI: Click here for additional data file.10.1107/S2056989021006381/ex2046sup7.pngHistogram showing the number of C...H-Br angles in hexacoordinated nitrosylrhenium complexes found in the database survey (section 5). DOI: 2075886CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Interlayer van der Waals and interhalogen interactions stabilize the packing.In the crystal, molecules are linked by C—H⋯O hydrogen bonds, forming layers parallel to the (001) plane. These layers of molecules are connected by C—H⋯π interactions along the  24H18Cl3NO3, the tetrahydrofuran rings adopt envelope conformations. In the crystal, C—H⋯O hydrogen bonds connect molecules, generating layers parallel to the (001) plane. These layers are connected along the c-axis direction by C—H⋯π interactions. The packing is further stabilized by interlayer van der Waals and interhalogen interactions. The most important contributions to the surface contacts are from H⋯H (36.8%), Cl⋯H/H⋯Cl (26.6%), C⋯H/H⋯C (18.8%) and O⋯H/H⋯O (11.3%) interactions, as concluded from a Hirshfeld surface analysis.In the title compound, C It was demonstrated that the products of the parallel [4 + 2] cycloaddition of two aryne moieties to both the furan fragments of the bis-diene system  prevails over the adduct (3) of the IMDAF reaction  in combination with other known reactions in a tandem or sequential manner is pursued for the construction of several important bicyclic or polycyclic compounds, including natural ones  to +1.4233 (blue) a.u. is shown in Fig. 7A and H16A, which highlight their functions as donors and/or acceptors. Fig. 8a) and those delineated into the major contacts: H⋯H  interactions are the major factor in the crystal packing together with Cl⋯H/H⋯Cl , C⋯H/H⋯C  and O⋯H/H⋯O  interactions representing the next highest contributions. The percentage contributions of other weak interactions are listed in Table 3In the crystal, hydrogen bonds of the C—H⋯O type link the molecules, generating layers parallel to the (001) plane Table 1 and 6 ▸.et al., 2016viz. 4,5-dibromo-2-[4-(trifluoromethyl)phenyl]hexahydro-3a,6-epoxyisoindol-1(4H)-one -yl]methyl}-2,3-dihydro-1H-isoindol-1-one -one -one -7a-chloro-2-[(4-nitrophenyl)sulfonyl]-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole -7a-chloro-6-methyl-2-[(4-nitrophenyl)sulfonyl]-1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole -7a-bromo-2-[(4-methylphenyl)sulfonyl]-1,2,3,6,7,7a-hexahydro-3a,6-epoxyiso-indole -7a-bromo-2-methylsulfonyl-1,2,3,6,7,7-hexahydro-3a,6-epoxyisoindole isoindole-5-carboxylate  and ac plane. Additionally, C—H⋯O hydrogen bonds form a C(6) chain, linking the molecules in the b-axis direction. In the crystal of ERIVIL, the molecules are connected into b-axis direction. In MIGTIG, the molecules are linked only by weak van der Waals interactions.In the crystal of IQOTOA, the asymmetric unit consists of two crystallographically independent molecules. In both molecules, the pyrrolidine and tetrahydrofuran rings adopt envelope conformations. In the crystal, molecules are linked in pairs by C— H⋯O hydrogen bonds. These pairs form a tetrameric supramolecular motif, leading to molecular layers parallel to the (100) plane formed by C— H⋯π and C—Br⋯π interactions. OMUTAU also crystallizes with two independent molecules in the asymmetric unit. In the central ring systems of both molecules, the tetrahydrofuran rings adopt envelope conformations, the pyrrolidine rings adopt twisted-envelope conformations and the six-membered ring is in a boat conformation. In both molecules, the nine-membered groups attached to the central ring system are essentially planar. In the crystal, strong intermolecular O—H⋯O hydrogen bonds and weak intermolecular C—H⋯O contacts link the molecules, forming a three-dimensional network. In addition, weak π–π stacking interactions between the pyrrolidine rings are observed. OMEMAX again crystallizes with two molecules in the asymmetric unit of the unit cell. In both molecules, the tetrahydrofuran rings adopt envelope conformations with the O atoms as the flaps and the pyrrolidine rings also adopt envelope conformations. In the crystal, molecules are linked by weak C—H⋯O hydrogen bonds, forming sheets lying parallel to the (001) plane. These sheets are connected only by weak van der Waals interactions. In the crystal of IMUBIE, the molecules are linked into dimers by pairs of C—H⋯O hydrogen bonds, thus generating N,N-bis(furan-2-ylmethyl)acetamide (0.0022 mol) dissolved in dry CH3CN (20 mL). Then an equivalent of 2-(trimethylsilyl)phenyl trifluoromethanesulfonate  was added to the solution under an argon atmosphere. The mixture was refluxed for 4 h (TLC control). After that, one more portion of 2-(trimethylsilyl)phenyl trifluoromethanesulfonate  and CsF  was added to the mixture, repeating all procedures again. After the mixture was cooled, CsF was filtered off through a thin layer of SiO2, and the resulting solution was concentrated under reduced pressure. The residue (brown oil) was separated using column chromatography on silica gel (a mixture EtOAc/hexane = 1/25 as eluent) to give compounds 1–3 in the ratio ∼30/25/45. Single crystals of compound 1 was obtained by slow crystallization from a hexane/EtOAc mixture.CsF  was added to 2,2,2-trichloro-1: white powder ; fR 0.50 ; m.p. 431.7–433.4 K. 1H NMR  δ 7.19–7.24 , 7.07 , 7.04 , 6.95–6.99 , 6.85 , 5.73 , 5.71 , 5.11 , 4.87 , 4.76 , 4.72 . 13C NMR  d 161.5, 150.2, 149.9, 148.7, 148.2, 145.2, 145.1, 143.3, 143.0, 125.5, 125.3, 125.2, 125.1, 120.5, 120.2, 120.0, 119.6, 94.1, 93.3, 92.1, 82.4, 82.2, 49.1, 45.6. IR νmax/cm−1 (tablet KBr): 2953, 2919, 1702, 1632, 1462, 1410, 1236. HRMS (ESI–TOF): calculated for C24H18Cl3NO4 [M + H]+ 473.0352; found 473.0358.Compound Uiso(H) = 1.2Ueq(C). Six reflections  I. DOI: 10.1107/S2056989021009907/yk2156Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021009907/yk2156Isup3.cmlSupporting information file. DOI: 2095762CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in calcium influx secondary to the upregulation of CaV1.2 channel subunits. Confocal imaging of norepinephrine (NE)-treated NRVMs revealed a hypertrophic response compared to untreated NRVMs. L-type CaV1.2 peak current density was increased 4-fold following a 24-h stimulation with NE. NE-treated NRVMs exhibited a significant upregulation of CaVα2δ1 and CaVβ3 protein levels without significant changes of CaVα1C and CaVβ2 protein levels. Pre-treatment with the β1-blocker metoprolol failed to inhibit hypertrophy or CaVβ3 upregulation whereas CaVα2δ1 protein levels were significantly reduced. NE promoted the phosphorylation of ERK 1/2, and the response was attenuated by the β1-blocker. U0126 pre-treatment suppressed NE-induced ERK1/2 phosphorylation but failed to attenuate hypertrophy. U0126 inhibition of ERK1/2 phosphorylation prevented NE-mediated upregulation of CaVα2δ1, whereas CaVβ3 protein levels remained elevated. Thus, β1-adrenergic receptor-mediated recruitment of the ERK1/2 plays a seminal role in the upregulation of CaVα2δ1 in NRVMs independent of the concomitant hypertrophic response. However, the upregulation of CaVβ3 protein levels may be directly dependent on the hypertrophic response of NRVMs.Intracellular Ca Severpression ,4,5. Carpression ,7,8. In pression ,7,8. Durpression ,7,8. Morpression ,7,8.2+ channels (LTCCs) mediate extracellular Ca2+ entry initiating Ca2+-induced Ca2+ release and triggering cardiac excitation-contraction coupling [Vα1 bound to the auxiliary subunits CaVβ, calmodulin, and CaVα2δ [Vα1C [Vβ2 [Vα2δ1 [Vα1C is the pore-forming subunit responsible for Ca2+ selectivity, activation and inactivation kinetics [Vβ is a cytoplasmic chaperone protein that supports the trafficking of CaV1.2 channels to the plasma membrane [Vα2δ1 is a large extracellular Glycosylphosphatidylinositol (GPI)-anchored protein that facilitates channel activation at physiological voltages and stimulates (≈5-fold) peak current density [Vα1C and CaVα2δ1 increased the peak current density of CaV1.2 [In ventricular cardiomyocytes, voltage-gated L-type Cacoupling ,9,10. LTd CaVα2δ ,12. In v2δ [Vα1C ,14, CaVβα1C [Vβ2 ,16, and 2 [Vα2δ1 ,18. CaVαkinetics . CaVβ ismembrane ,21,22. C density ,24,25. If CaV1.2 .1-adrenergic receptor activation increases voltage-gated L-type Ca2+ currents via protein kinase A phosphorylation which releases the Rad-mediated inhibition through a likely reduction in the affinity of Rad with the membrane and the CaVβ-subunit of the channel [2+ overload remains unresolved. Therefore, the present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in Ca2+ influx secondary to the upregulation in the protein expression of CaV1.2 channel subunits.Numerous studies have identified the sympathetic system as an essential homeostatic mechanism providing inotropic support, whereas chronic activation contributes in part to the progression of cardiac hypertrophy. Acute sympathetic stimulation of ventricular cardiomyocytes via the β channel . The relThe use and care of laboratory rats were according to the Canadian Council for Animal Care and approved by the Animal Care Committee of the Montreal Heart Institute.2 in DMEM-low glucose  supplemented with 7% heat-inactivated FBS and 1% penicillin-streptomycin for 24 h, subsequently washed and maintained in DMEM-low glucose containing insulin (5 μg/mL), transferrin (5 μg/mL), and selenium (5 ng/mL)  for 24 h prior to the experimental protocol. In all experiments, neonatal rat ventricular cardiomyocytes were plated at a density of 400–500 cells/mm2.Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 1-day old Sprague-Dawley rat pups (sacrificed by decapitation)  as previously described ,27,28. VCardiomyocytes were treated with 1 µM norepinephrine (NE) for 1, 4, 16, 24, or 48 h. The untreated cardiomyocytes were subjected to the same procedure and plating duration as norepinephrine-treated cardiomyocytes. In parallel experiments, neonatal cardiomyocytes were pre-treated with 10 µM U0126  or 100 nM metoprolol tartrate  for 30 min to 1 h prior to the addition of 1 µM NE for 24 h.Vα2δ1 , and rabbit anti-CaVα1C . Secondary antibodies employed include goat anti-mouse coupled to Alexa 555  and donkey anti-rabbit coupled to Alexa 488 . The nucleus was identified with 4′,6′-diamidino-2-phenylindole  staining, and 4′,6-diamidino-2-phenylindole DAPI (1:800). Images were captured by confocal microscope with 20× or 63× objective. Images were analyzed using Zeiss LSM image software. The cross-sectional area (µm2) of 250 Troponin-T+ mononucleated cardiomyocytes from 5 different images per condition was determined using Zeiss LSM image software. For the colocalization analysis, wheat germ agglutinin (WGA) antibody  was added to cardiomyocytes prior to fixation.Neonatal rat ventricular cardiomyocytes (NRVMs) were plated on glass coverslips coated with poly-D-lysine in 12-well plates. Cardiomyocytes were fixed with 2% paraformaldehyde for 20 min with 2% paraformaldehyde. Immunofluorescence was performed as previously described . PrimaryVα2δ1 , rabbit CaVα1C , rabbit CaVβ2 , rabbit CaVβ3 , rabbit Phospho-ERK 1/2 , Total ERK 1/2  and rabbit GAPDH . Membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with secondary anti-rabbit antibody  for 2 h at room temperature. Signal was detected using enhanced chemiluminescence (ECL) substrate.Protein lysates of neonatal rat ventricular cells were prepared and subjected to SDS-electrophoresis, as previously described . Lysates2, 1 MgCl2, and 10 HEPES, titrated to pH 7.4 with HCI. The measurements were performed at room temperature (22–25 °C).Whole-cell voltage-clamp recordings were performed in isolated NRVMs. Patch-clamp experiments were carried out with the Axopatch 200-B amplifier . PClamp software Clampex 10.4 coupled to a Digidata 1440A acquisition system  was used for on-line data acquisition and analysis. Electrodes were filled with a solution containing (in mM) 140 CsCl, 0.6 NaGTP, 3 MgATP, and 10 EGTA, 10 HEPES, titrated to pH 7.4 with KOH. Pipette resistance ranged from 3 to 5 megohms. The bath solution contained (in mM) 135 NMDG, 20 tetraethylammonium chloride, 2 CaCl+ channels, Ca2+ currents were elicited from a holding potential of −80 mV and were depolarized to potentials ranging from −80 to 50 mV in 5 mV increments lasting 450 ms for each step (protocol shown in the inset above the current traces). Ca2+ current densities (pA/pF) were obtained by dividing the peak currents by the cell capacitance. Average I-V curves were obtained by plotting peak current densities as a function of applied voltage. The I-V relationships were fitted to a Boltzmann equation. Patch-clamp data were analyzed using Clamp fit software 10.4 (Molecular Devices), Microsoft Excel 2016 , and Origin 2020 .Following a 40 ms prepulse to −40 mV to inactivate Nap < 0.05 was considered statistically significant.Data were presented as the mean ± S.E.M., and n represents the number of rat litters employed. Data were evaluated by one-way ANOVA analysis followed by Dunnett’s multiple comparison test or Tukey’s multiple comparison test (GraphPad). A value of V1.2 peak current density. Whole-cell Ca2+ current traces were detected in untreated NRVMs  (p < 0.01) four fold increase in peak current density was detected    in response to 1 µM norepinephrine (NE) was examined. As compared to untreated NRVMs, a significant hypertrophic response as depicted by the increase in cell surface area was detected after a 24-h stimulation with NE, and hypertrophy persisted after 48 h A,B. In p, N = 2) A,B. In h, N = 2) A,B. By c = 0.13) C.Vα2δ1 protein levels at 24 h and remained elevated at 48 h as compared to untreated NRVMs . NRVMs were stained with cardiac troponin-T (staining not shown), CaVα1C subunit, wheat germ-agglutinin (WGA), or 4′,6-diamidino-2-phenylindole . CaVα1C localization was determined by Pearson’s coefficient assessing the convergence of the immunofluorescent signal between WGA/CaVα1C and DAPI/CaVα1C. Vα1C (green). In untreated NRVMs, CaVα1C staining was detected predominantly on the plasma membrane and perinuclear region  as compared to untreated NRVMs   A,C.Vα1C (green). The co-staining of NRVMs with DAPI and CaVα1C confirmed the perinuclear localization  of the pore-forming subunit  and was similar to that observed in untreated cardiomyocytes  ( subunit B. In NE- > 0.05) B,D.Vα2δ1 was localized at the plasma membrane of neonatal mouse cardiomyocytes [Vα2δ1 protein levels secondary to NE-induced cardiac hypertrophy was associated with a greater translocation to the plasma membrane. Therefore, the colocalization of CaVα2δ1 with WGA or DAPI was examined. Vα2δ1 (green). In NRVMs treated with 1 µM NE for 24 or 48 h, CaVα2δ1 localization at the plasma membrane was not altered  when compared to untreated NRVMs  (Previous studies reported that Camyocytes . Immunof > 0.05) A,C. The Vα2δ1 (green). The co-staining of NRVMs with DAPI and CaVα2δ1 in untreated NRVMs revealed a perinuclear and nuclear signal  of the calcium subunit  ) with NRVMs treated with 1 µM NE for 24 or 48 h ; p > 0.05 (Vα2δ1 (green). The co-staining with DAPI and CaVα2δ1 in untreated NRVMs revealed the presence of the subunit in perinuclear and nuclear regions, depicted by a yellow signal    ( ~ 0.51) B. As seep > 0.05 A,C. Figu ~ 0.51) B. CaVα2δ; n = 3) B,D. In N; n = 3) B,D.1-adrenergic receptor antagonist metoprolol (100 nM) failed to inhibit NE-mediated hypertrophy , NE-mediated ERK1/2 phosphorylation was inhibited  were treated with norepinephrine as numerous studies have previously established a hypertrophic role of the sympathetic neurotransmitter. As demonstrated in previous studies, the exposure of NRVMs to sympathetic stimulation for 24 and 48 h led to a significant increase in the cell surface area as compared to untreated NRVMs. In parallel, L-type CaV1.2 peak current density was significantly elevated in NRVMs secondary to NE-induced cardiomyocyte hypertrophy. These data provided the impetus to assess the individual role of the subunits forming the oligomeric CaV1.2 channel in the increased Ca2+ influx in NE-induced hypertrophied NRVMs.Numerous in vitro and in vivo studies have delineated the role of calcium-dependent signaling events linking various stimuli to ventricular cardiomyocyte hypertrophy ,30,31. H2+ channels in ventricular cardiomyocytes in response to acute sympathetic stimulation occurs predominantly via β-adrenergic receptor-mediated recruitment of protein kinase A and subsequent channel phosphorylation [2+ channel activity. The increase in the L-type CaV1.2 peak current density in NE-treated NRVMs was associated with a significant upregulation of CaVα2δ1 with a more modest increase in the protein expression of CaVβ3. In contrast, the protein levels of the pore-forming CaVα1C and the accessory CaVβ2 were unchanged. Previous work from our lab revealed that co-expression of CaVα1C and CaVα2δ1 subunits upregulated by 5- to 10-fold the peak current density and facilitated the opening of the L-type CaV1.2 activity at physiological voltages [Vβ3 protein levels in NE-induced hypertrophy of NRVMs was reported to be insufficient to promote on its own a change in the activity of CaV1.2 [Vα2δ1 protein levels in NE-treated NRVMs was blunted by pre-treatment with the selective β1-blocker metoprolol, whereas CaVβ3 subunit upregulation remained unchanged. Moreover, metoprolol did not inhibit NE-mediated hypertrophy, which was in part consistent with the predominant role of the α1-adrenergic receptor in cardiomyocyte hypertrophy [1-adrenergic receptor-mediated upregulation of CaVα2δ1 protein levels in response to NE may have contributed in part to increased L-type Ca2+ channel activity independent of the concomitant hypertrophic response. By contrast, upregulation of CaVβ3 protein levels may be directly dependent on the hypertrophic response of NRVMs secondary to sympathetic stimulation. These data are supportive of β1-adrenergic receptor-mediated NE-induced upregulation of CaVα2δ1, but other regulators of adrenergic receptors need to be added to support the conclusion.Activation of voltage-gated L-type Carylation . The prevoltages . The morf CaV1.2 . The uprertrophy . CollectVα2δ1 protein expression was not accompanied by a significant change in its plasma membrane localization. Immunofluorescence confocal images of untreated and NE-treated NRVMs co-stained with WGA and CaVα2δ1 revealed that localization of the subunit at the plasma membrane was similar. Moreover, the co-staining of DAPI and CaVα2δ1 in perinuclear and nuclear regions was not significantly modified in NE-treated NRVMs. Similarly, we failed to observe any significant change in the localization of CaVα1C at the plasma membrane in NE-hypertrophied NRVMs. It remains to be seen if the perinuclear/nuclear distribution of CaVα2δ1 and CaVα1C is exclusive to NRVMs or is conserved in adult cardiomyocytes after postnatal development.The upregulation of the CaVα2δ1 protein levels via recruitment of the ras/MEK/ERK1/2 signaling pathway [1-adrenergic receptor is known to promote ERK1/2 phosphorylation [1-adrenergic receptor antagonist metoprolol suppressed NE-mediated ERK1/2 phosphorylation. Furthermore, pharmacological inhibition of the upstream activator MEK with U0126 suppressed NE-mediated ERK1/2 phosphorylation and the concomitant upregulation of CaVα2δ1 protein levels. By contrast, U0126 failed to inhibit NE-mediated NRVM hypertrophy and upregulation of CaVβ3 protein levels. The absence of an anti-hypertrophic effect of U0126 after NE treatment of NRVMs was consistent with previous data demonstrating that recruitment of the ERK1/2 signaling pathway alone was insufficient to promote cardiomyocyte hypertrophy in response to various stimuli [A previous study reported that EGF stimulation of GH3 pituitary cells increased Ca pathway . In NRVMrylation . NE trea stimuli .2+ influx via activation of the L-type CaV1.2 channel after sympathetic discharge [2+ channel activity. The latter response required β1-adrenergic receptor-mediated recruitment of the tyrosine kinase ERK1/2 translating to the increased expression of the CaV1.2 auxiliary subunit, CaVα2δ1. β1-adrenergic receptor-mediated upregulation of the CaVα2δ1 subunit was independent of the hypertrophic response. Upregulation in the protein expression of the CaVα2δ1 subunit secondary to sympathetic hyperactivity may hence contribute to intracellular Ca2+ overload with or without hypertrophy [The present study has revealed that in addition to the acute increase in Caischarge , chronicertrophy .


The complete polyamine dication is visible in the electron density and assumes a position that allows for hydrogen-bond interactions with three Z-DNA duplexes. The K+ cation occupies a mixed K+/H2O site and forms an irregular coordination sphere interacting with water molecules and the O atoms of two phosphate groups of two Z-DNA molecules, with some of the ligands present at fractional occupancy.An ultra-high-resolution crystal structure of Z-DNA with the sequence d(CGCGCG) 2/putrescine(2+)/K+ complex has been determined at 0.60 Å resolution. Stereochemical restraints were used only for the putrescinium dication, and 23 bonds and 18 angles of the Z-DNA nucleotides with dual conformation. The N atoms of the putrescine(2+) dication form three direct hydrogen bonds with the N7_G atoms of three different Z-DNA molecules, plus three water-mediated hydrogen bonds with cytosine, guanine and phosphate acceptors. A unique potassium cation was also unambiguously identified in the structure, albeit at a ∼0.5 occupation site shared with a water molecule, providing the first example of such a complex with Z-DNA. The K+ cation has coordination number of eight and an irregular coordination sphere, formed by four water molecules and four O atoms from two phosphate groups of the Z-DNA, including ligands present at fractional occupancy. The structural disorder of the Z-DNA duplex is manifested by the presence of alternate conformations along the DNA backbone. Comparison of the position and interactions of putrescine(2+) in the present structure with other ultra-high-resolution structures of Z-DNA in complexes with Mn2+ and Zn2+ ions shows that the dicationic putrescinium moiety can effectively substitute these metal ions for stabilization of Z-type DNA duplexes. Furthermore, this comparison also suggests that the spermine(4+) tetracation has a higher affinity for Z-DNA than K+.The X-ray crystal structure of the d(CGCGCG) Put2+ and Cad2+ are the two most common biogenic diamines in bacteria. For example, at a concentration of 10–30 mM, putrescine is the predominant polyamine in Escherichia coli  are involved in maintaining the cell-envelope integrity of bacteria. They are also crucial to the virulence phenotype of many bacterial pathogens  are important resources for viruses, which evolved mechanisms to maintain, enhance or manipulate polyamine(n+) metabolism to support viral infection . Polyamines(n+) are important factors stabilizing nucleic acids and stimulating their replication; only ∼7–10% of the total cell content exists as free polyamines(n+), the vast majority remaining in association with nucleic acids  and metal polycations (even at low concentration) can stimulate B to Z conversion of DNA and promote DNA condensation  and metal cations can interact with Z-DNA molecules in one of two different modes. Polyamine(n+) cations are found to bind to the O atoms of different phosphate groups either directly or through water-mediated bridges. In the second mode, the cations are coordinated simultaneously by the guanine bases from different helices and also by the O atoms of dual-conformation phosphate groups , Spd3+ (2elg and 293d), N-(2-aminoethyl)-1,4-diaminobutane (292d), thermospermine(4+) (336d), trientine(4+) (1dj6) and N1-{2-[2-(2-aminoethylamino)ethylamino]ethyl}ethane-1,2-diamine (2ie1) are also deposited in the PDB. Besides the Mg2+ cation, there are PDB structures of Z-DNA or Z-DNA-RNA  cations, including Ba2+, Mn2+, Cu2+, Zn2+, Cr3+, Co3+ and Ru3+. There is currently no structure, however, of a Z-DNA/K+ complex.Among all Z-DNA 2.2.1.et al., 1998et al., 2013M water solution of the DNA oligonucleotide with the self-complementary sequence d(CGCGCG) was heated at 338 K for 10 min and then annealed slowly to room temperature overnight. Single crystals of the d(CGCGCG)2/Put2+/K+ complex were grown at 292 K by the hanging-drop vapour-diffusion method by mixing a 2 µl nucleic acid solution and a 2 µl precipitating solution consisting of 10%(v/v) (+/−)-2-methyl-2,4-pentanediol (MPD), 40 mM sodium cacodylate pH 6.0, 80 mM KCl, 12 mM NaCl and 14 mM putrescinium dichloride. The drops were equilibrated against 0.5 ml 80%(v/v) MPD. Crystals appeared within one week and grew to dimensions of 0.3 mm × 0.1 mm × 0.1 mm.The methods of synthesis, deprotection and purification of the oligodeoxynucleotide have been described previously 2/Spm4+/Mn2+ complex  of K and P atoms are, respectively, 0.236, 0.286 and 0.095, 0.104 electron units , the imaginary component of the anomalous scattering  as described by Kowiel et al. .Figures presenting the model and electron density were prepared with 2.4.7atg. Raw X-ray diffraction images were deposited in the Integrated Resource for Reproducibility in Macromolecular Crystallography repository  described by Brzezinski et al. (2011The estimated standard uncertainties (e.s.u.) of the fully occupied DNA atomic positions in the structure are in the range 0.003–0.01 Å for C atoms, 0.003–0.007 Å for N atoms, 0.003–0.009 Å for O atoms and 0.001–0.002 Å for P atoms. The e.s.u. values for the full-occupancy covalent bonds are ∼0.005, ∼0.004, ∼0.004 and ∼0.003 Å for C—C, C—O, C—N and P—O, respectively. The agreement with stereochemical standards (r.m.s.d.) is 0.010 Å for bond lengths and 1.56° for bond angles. These results are comparable in terms of accuracy and precision with the record-setting model of Z-DNA  per CG/GC tandem of base pairs is −60°. Other average base-pair and base-pair-step parameters are as follows: helical rise 4.37 Å, inclination (η) 14.55°, tip 2.6°, tilt 0.44°, roll −3.32°, shift −0.02 Å, slide 2.77 Å and rise 3.45 Å. Comparison of these helical parameters and base-pair geometries for the present and previously described Z-DNA complexes shows that they are within the range typical for Z-DNA duplexes endo and C1′-exo sugar puckers with pseudorotation (P) angles of 162.0 (7) and 138.2 (6)°, respectively. Differences in sugar pucker are also observed at G4, where conformation I is C3′-endo [P = 20.2 (7)°] and conformation II is C4′-exo [P = 38 (1)°]. Moreover, the mFo–DFc electron-density maps indicate three additional discrete positions (peaks) for the phosphate atoms at the C3 (6.7σ), C9 (6.0σ) and C11 (6.7σ) nucleotides. However, additional phosphate groups placed at these peaks refined with an occupancy below 0.20. Therefore, no atoms were modelled to interpret the peaks near the P_C3, P_C9 and P_C11 atoms. No alternative conformations were observed for the base moieties.The 5′-d(CGCGCG) nucleotides of chain A in the asymmetric unit are numbered 1–6 and the complementary 3′-d(GCGCGC) nucleotides of chain B are numbered 12–7. There are only five internucleotide phosphodiester groups on each strand, which are numbered, respectively, P2–P6 and P12–P8. Alternative conformations are clearly visible in the electron-density map and are designated as I/II at the C3–G4, G4–C5 and C5–G6 internucleotide phosphate linkages, whose refined occupancies converged at 0.687 (8)/0.313 (8), 0.710 (4)/0.290 (4) and 0.650 (12)/0.350 (12), respectively. The major (I) and minor (II) conformations of C3 have C2′-endo/C3′-endo pucker of the pyrimidine/purine nucleotides, as is typical for Z-DNA, but all assume the C2′-endo conformation. The ZII conformation of the phosphate group can be assigned only to G4(I) with ζ = 64.6° [ζ is a backbone torsion angle defined as: C3′—O3′—P(i + 1)—O5′(i + 1)]. This ZII conformation is stabilized by a water-mediated OP2(I)_G4⋯Wat81⋯N2_G4 hydrogen bond.In the present complex, the sugars at the 3′-termini do not have the alternating C2′-3.3.gauche−–trans–gauche+ conformation, with the following torsion angles: −72.5 (7), 172.9 (5) and 71.0 (8)°. The Put2+ dication is involved in direct hydrogen-bond contacts only with the guanine N7 atoms of three DNA duplexes  Å and N1_Put2+⋯N7_G10i = 2.755 (6) Å [symmetry code: (i) x − y + z + 1]. The N2 atom is hydrogen bonded to N7_G6ii at a distance of 2.839 (7) Å . The structure of Z-DNA with Dap2+ (PDB ID 2f8w) indicates that this one-carbon-link-shorter polyamine interacts only with the phosphate groups of the Z-DNA  exhibits a preference for binding with Z-DNA phosphates only .Although the crystallization systems for all the Z-DNA complexes presented in our previous studies (Drozdzal gauche−–trans–gauche+ conformation, while in the remaining cases it is all-trans (Woo et al., 1979et al., 1986et al., 2007The putrescinium dication has been characterized in several crystal structures of its salts and in some of them it has the Our study provides insight into the effectiveness and competition of polyamine and metal cations for interactions with Z-DNA, and confirms that even a simple diamine can adopt different conformations and consequently enter into a variety of interactions with biomacromolecular partners.+ ion confirms previous research findings suggesting that most observed monovalent cation sites in DNA crystals are partially occupied positions (Tereshko et al., 2001et al., 1997et al., 1998The partial occupancy of the K10.1107/S2052520621002663/aw5052sup1.pdfPDB Validation Report. DOI: https://dx.doi.org/10.18430/m37atgRaw X-ray diffraction data:


This complex consists of discrete binuclear molecules with a {Cu2(μ2-Cl)2} core.The title compound, [Cu 2Cl4(C5H10N2O2)2(CH3CN)2] or [Cu2(μ2-Cl)2(CH3—Pz-COOCH2CH3)2Cl2(CH3CN)2], was synthesized using a one-pot reaction of copper powder, copper(II) chloride dihydrate and ethyl 5-methyl-1H-pyrazole-3-carboxylate (CH3—Pz-COOCH2CH3) in acetonitrile under ambient conditions. This complex consists of discrete binuclear molecules with a {Cu2(μ2-Cl)2} core, in which the Cu⋯Cu distance is 3.8002 (7) Å. The pyrazole-based ligands are bidentate coordinated, leading to the formation of two five-membered chelate rings. The coordination geometry of both copper atoms (ON2Cl3) can be described as distorted octahedral on account of the acetonitrile coordination. A Hirshfeld surface analysis suggests that the most important contributions to the surface contacts are from H⋯H (40%), H⋯Cl/Cl⋯H (24.3%), H⋯O/O⋯H (11.8%), H⋯C/C⋯H (9.2%) and H⋯N/N⋯H (8.3%) interactions.The title compound, [Cu The copper atom has a distorted octahedral coordination environment formed by three chlorine atoms, one nitrogen atom of the acetonitrile molecule and two atoms of the unsymmetrical pyrazole ligand – the pyridine-like N1 atom and atom O 1 of the ester substituent in position 3 of the pyrazole ring. The bidentate coordination of the pyrazole ligand leads to the formation of a five-membered chelate ring. The atoms in the ring deviate only slightly from planarity . Both the copper atoms and the bridging chlorine atoms lie in the same plane without deviations from planarity. The intermetallic distance in the dimer unit is 3.8002 (7) Å while the chlorine–chlorine separation in the four-membered bimetallic cycle is 3.5894 (15) Å.The title compound Fig. 1 is a binH-pyrazole-3-carboxylate (green) is presented in Fig. 2OLEX2 software  Å and Cl⋯Cl = 3.5364 (11) Å for the methyl analogue]. The molecular structure is stabilized by intramolecular N—H⋯Cl and C—H⋯Cl hydrogen bonds (Table 1An overlay of the asymmetric units of the structures of the title compound (red) and a similar complex with methyl 5-methyl-1s Table 1.2(μ2-Cl)2} core, which form a planar bimetallic ring. The four-membered Cu1/Cl1/Cu1i/Cl1i planes of the bimetallic rings are situated perpendicular to the b axis, while the chelate ring planes are located approximately parallel. No intermolecular hydrogen bonds were identified in the crystal structure. The minimum separation between the Cl atoms of neighbouring molecules inside one unit cell is 4.4013 (13) Å for Cl1i and Cl1ii  while the minimum distance between two copper atoms is 7.6498 (3) Å for Cu1 and Cu1ii.The crystal packing of the title compound Fig. 3 consistsCrystal Explorer 17.5  to 1.1926 (blue) a.u. The pale-red spots in Fig. 4dnorm values on the surface corresponding to the interactions described above. The Hirshfeld surfaces mapped over dnorm are shown for the H⋯H, H⋯Cl/Cl⋯H, H⋯O/O⋯H, H⋯C/C⋯H and H⋯N/N⋯H contacts, the overall two-dimensional fingerprint plot and the decomposed two-dimensional fingerprint plots are given in Fig. 53 group of the acetonitrile molecule. For the title compound, the most significant contributions to the overall crystal packing are from H⋯H (40%), H⋯Cl/Cl⋯H (24.3%), H⋯O/O⋯H (11.8%), H⋯C/C⋯H (9.2%) and H⋯N/N⋯H (8.3%) contacts. The small contribution of the other weak intermolecular C⋯C (2.9%), C⋯O/O⋯C (2.1%), C⋯N/N⋯C (0.8%), C⋯Cl/Cl⋯C (0.3%), O⋯N/N⋯O (0.3%) and Cl⋯Cl (0.1%) contacts has a negligible effect on the packing. In addition, quantitative physical properties of the Hirshfeld surface for the title compound were obtained, such as molecular volume (657.89 Å3), surface area (571.56 Å2), globularity (0.640), as well as asphericity (0.147).The Hirshfeld surface analysis and the associated two-dimensional fingerprint plots were performed using et al., 2016H-pyrazole-3-carboxylate [UMUXEI 2(CH3-Pz-COOCH2CH3)2Cl2(CH3CN)2] was synthesized at room temperature by the oxidative dissolution method by the addition of a copper powder  and copper(II) chloride dihydrate  mixture to an acetonitrile (9 ml) solution of ethyl 5-methyl-1H-pyrazole-3-carboxylate . The mixture was stirred without heating for three h with free air access until dissolution of the copper powder and a green precipitate of the product was obtained. The precipitate was filtered off and re-dissolved in acetonitrile. Green crystals suitable for X-ray analysis were obtained by slow evaporation of the solvent. The IR spectra of the starting pyrazole ligand and the obtained green crystals of the title coordination compound are given in the supporting information.[CuUiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). N-bound H atoms were refined with Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021010653/zq2267sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021010653/zq2267Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021010653/zq2267Isup3.cdxSupporting information file. DOI: 10.1107/S2056989021010653/zq2267sup4.txtIR spectrum of the title compound. DOI: 10.1107/S2056989021010653/zq2267sup5.txtIR spectrum of the free ligand. DOI: 2115608CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information: