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1.
7-Methylpterin and 7-methyllumizine were isolated and identified in extracts of methanogenic bacteria which had been extracted in air with ethanol-water. Ethanol-water preparations of cells extracted under nitrogen or hydrogen were devoid of these compounds. Extracts of cells obtained in the presence of air also had an increased amount of a complex arylamine which, on acid hydrolysis, gave 1 mol each of phosphate, 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane, and alpha-hydroxyglutaric acid. Gas chromatography-mass spectrometry was used to identify the 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane as its tetratrimethylsilyl derivative and the alpha-hydroxyglutaric acid as the n-butyl ester derivative of its gamma-lactone. When exposed to air, extracts of cells prepared in the absence of air produced 6-acetyl-7-methylpterin and 7-methylxanthopterin in addition to 7-methylpterin and 7-methyllumizine. It is concluded that these compounds are derived from the oxidative cleavage of the tetrahydromethanopterin, which is present in these bacteria, by a series of reactions analogous to those known to occur in the oxidative cleavage of tetrahydrofolic acid.  相似文献   

2.
The established pathway for the last two steps in purine biosynthesis, the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) to IMP, is known to utilize 10-formyl-tetrahydrofolate as the required C1 donor cofactor. The biosynthetic conversion of ZMP to IMP in three members of the domain Archaea, Methanobacterium thermoautotrophicum deltaH, M. thermoautotrophicum Marburg, and Sulfolobus solfataricus, however, has been demonstrated to occur with only formate and ATP serving as cofactors. Thus, in these archaea, which use methanopterin (MPT) or another modified folate in place of folate as the C1 carrier coenzyme, neither folate nor a modified folate serves as a cofactor for this biosynthetic transformation. It is concluded that archaea, which function with modified folates such as MPT, are able to carry out purine biosynthesis without the involvement of folates or modified folates.  相似文献   

3.
Analyses were made of the structures and levels of folates and modified folates present in extremely thermophilic bacteria. These procedures involved the chemical analysis of products resulting from the oxidative cleavage of the 6-substituted, folatelike tetrahydropterins present in the cells. Air-oxidized cell extracts of extreme thermophiles from two members of the archaebacterial order Thermococcales, Thermococcus celer and Pyrococcus furiosus, contained only 7-methylpterin, indicating that these cells contain a modified folate with a methylated pterin. Cell extracts also contained 6-acetyl-7-methyl-7,8-dihydropterin, another product derived from the oxidative cleavage of a dimethylated folate, demonstrating that both the C-7 and C-9 carbons of the pterin were methylated. Extracts, however, contained neither p-aminobenzoylpolyglutamates nor methaniline, the oxidative cleavage products of folates and methanopterin, respectively, indicating that they contain a previously undescribed C1 carrier(s). On the basis of the level of the 7-methylpterin isolated, the levels of modified folate were 2 to 10 times higher than those typically found in mesophilic bacteria and 10 to 100 times less than the level of methanopterin found in the methanogenic bacteria. Oxidized cell extracts of Sulfolobus spp. of the archaebacterial order Sulfolobales contained only pterin, and, like members of the order Thermococcales, they contained neither-p-aminobenzoylpolyglutamates nor methaniline. Oxidized cell extracts of the extreme thermophiles Pyrobaculum sp. strain H10 and Pyrodictium occultum, from the archaebacterial orders Thermoproteales and Pyrodictiales, respectively, and Thermotoga maritima from the eubacterial order Thermotogales, contained pterin and p-aminobenzoylpolyglutamates, indicating that these cells contained unmodified folates. The levels of p-aminobenzoylpolyglutamates in these archaebacterial cell extracts indicate that the folates were present in the cells at levels 4 to 10 times higher than generally found in those mesophilic eubacteria which do not folates in energy metabolism. The levels and chain lengths of the of p-aminobenzoylpolyglutamates present in Thermotoga maritima were typical of those found in mesophilic eubacteria.  相似文献   

4.
Biosynthesis of methanopterin   总被引:6,自引:0,他引:6  
R H White 《Biochemistry》1990,29(22):5397-5404
The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. [methylene-2H]-6-(Hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from [methyl-2H3]methionine, as confirmed by the incorporation of two C2H3 groups into 6-ethyl-7-methylpterin, a pterin-containing fragment derived from methanopterin. Cells grown in the presence of [methylene-2H]-6-(hydroxymethyl)pterin, [ethyl-2H4]-6-[1 (RS)-hydroxyethyl]pterin, [methyl-2H3]-6- (hydroxymethyl)-7-methylpterin, [ethyl-2H4, methyl-2H3]-6-[1 (RS)-hydroxyethyl]-7-methylpterin, and [1-ethyl-3H]-6-[1 (RS)-hydroxyethyl]-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic [methylene-3H]-7,8-H2-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H2-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.  相似文献   

5.
Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.  相似文献   

6.
Biosynthesis of the 7-methylated pterin of methanopterin.   总被引:4,自引:3,他引:1       下载免费PDF全文
The incorporation of [15N]glycine and [U-methyl-2H]methionine into methanopterin by growing cells of a methanogenic bacterium was measured to establish the biosynthetic route of the methylated pterin in the structure. The tetrahydromethanopterin produced by the cells was oxidatively cleaved to produce 7-methylpterin, and the amount of label incorporated into this pterin was measured by gas chromatography-mass spectrometry of the ditrimethylsilyl derivative of this compound. Approximately 27% of the 7-methylpterin and the guanine present in the cell was derived from the fed [15N]glycine. [U-methyl-2H]methionine was incorporated with the initial retention of all three deuteriums. These results are consistent with the biosynthesis of the pterin of methanopterin originating from GTP and its 7-methyl group arising from the methyl group of methionine.  相似文献   

7.
Evidence for folate-salvage reactions in plants   总被引:3,自引:1,他引:2  
Folates in vivo undergo oxidative cleavage, giving pterin and p -aminobenzoylglutamate ( p ABAGlu) moieties. These breakdown products are excreted in animals, but their fate is unclear in microorganisms and unknown in plants. As indirect evidence from this and previous studies strongly suggests that plants can have high folate-breakdown rates (approximately 10% per day), salvage of the cleavage products seems likely. Four sets of observations support this possibility. First, cleavage products do not normally accumulate: pools of p ABAGlu (including its polyglutamyl forms) are equivalent to, at most, 4–14% of typical total folate pools in Arabidopsis thaliana , Lycopersicon esculentum and Pisum sativum tissues. Pools of the pterin oxidation end-product pterin-6-carboxylate are, likewise, fairly small (3–37%) relative to total folate pools. Second, little p ABAGlu built up in A. thaliana plantlets when net folate breakdown was induced by blocking folate synthesis with sulfanilamide. Third, A. thaliana and L. esculentum tissues readily converted supplied breakdown products to folate synthesis precursors: p ABAGlu was hydrolysed to p -aminobenzoate and glutamate, and dihydropterin-6-aldehyde was reduced to 6-hydroxymethyldihydropterin. Fourth, both these reactions were detected in vitro ; the reduction used NADPH as cofactor. An alternative salvage route for p ABAGlu, direct reincorporation into dihydrofolate via the action of dihydropteroate synthase, appears implausible from the properties of this enzyme. We conclude that plants are excellent organisms in which to explore the biochemistry of folate salvage.  相似文献   

8.
Identification of a novel tatiopterin derivative in Methanogenium tationis   总被引:2,自引:0,他引:2  
Recently, a novel pterin has been isolated from Methanogenium tationis. This pterin derivative, which was called tatiopterin, was characterized as a methanopterin-like structure with an additional aspartyl and glutamyl group in the side chain and with a 7-proton instead of a 7-methyl group in the pterin moiety. The sequence of the aspartyl and glutamyl group remained unsolved. In this study, a novel pterin was purified from Mg.tationis and analyzed by 600 MHz 1H-NMR spectroscopy and fast atom bombardment-mass spectroscopy. This pterin was found to be an aspartyl derivative of methanopterin with a 7-proton in the pterin part of the molecule. No glutamyl group could be detected. Apparently, Mg.tationis is able to synthesize two types of tatiopterin derivatives. For these cofactors the trivial names 'tatiopterin-0' (lacking a glutamyl group) and 'tatiopterin-I' (containing one glutamyl group) are introduced here.  相似文献   

9.
Methanopterin and methanogenic bacteria   总被引:2,自引:0,他引:2  
Methanogenic bacteria comprise a selected group of microorganisms that derive their energy for growth from the hydrogen-dependent reduction of CO2 to methane or the disproportionation of reduced one-carbon compounds and acetate to CO2 and methane. In the reduction and oxidation steps at the formyl, hydroxymethyl and methyl level the one-carbon unit remains bound to the reduced form of methanopterin, a pterin derivative typical of methanogenic bacteria. In addition, the reduced methanopterin, 5,6,7,8-tetrahydromethanopterin, is involved in a number of anabolic reactions. Methanopterin is structurally and functionally the counterpart of folic acid found in other organisms. In this review the occurrence and properties of methanopterin and its derivatives, as well as the biosynthesis and the role in the different catabolic and anabolic reactions are discussed against the background of folic acid biochemistry.  相似文献   

10.
The biosynthesis of dTMP has been studied in cell extracts of two different members of the domain Archaea, Methanosarcina thermophila and Sulfolobus solfataricus. In M. thermophila, the dTMP was formed from dUMP and [methylene-2H2]-5,10-methylenetetrahydrosarcinapterin generated in situ from added [methylene-2H2] formaldehyde and the tetrahydrosarcinapterin present in the cell extract. In S. solfataricus, the 5,10-methyl-enetetrahydro derivative of a synthetic fragment of sulfopterin, the modified folate present in these cells, served as the C1 donor. These data indicate that the Archaea thymidylate synthases carry out the same basic reaction which occurs in other organisms but use the 5,10-methylenetetrahydro derivatives of modified folates as C1 donors.  相似文献   

11.
The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.  相似文献   

12.
Tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H4biopterin, suggesting that the natural cofactor has an additional, as yet unknown catalytic function. Here we show that the 5-methyl analog of H4biopterin, which does not react with O2, is a functionally active pterin cofactor of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective concentrations of approximately 0.2 microM and 10 mM 5-methyl-H4biopterin. Thus, the affinity of the 5-methyl compound was 3 orders of magnitude lower than that of the natural cofactor, allowing the direct demonstration of the functional anticooperativity of the two pterin binding sites of dimeric NOS. In contrast to H4biopterin, which inactivates nitric oxide (NO) through nonenzymatic superoxide formation, up to 1 mM of the 5-methyl derivative did not consume O2 and had no effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the detection of enzymatic NO formation in the absence of superoxide or NO scavengers. These results unequivocally identify free NO as a NOS product and indicate that reductive O2 activation by the pterin cofactor is not essential to NO biosynthesis.  相似文献   

13.
Dihydropterins are intermediates of folate synthesis and products of folate breakdown that are readily oxidized to their aromatic forms. In trypanosomatid parasites, reduction of such oxidized pterins is crucial for pterin and folate salvage. We therefore sought evidence for this reaction in plants. Three lines of evidence indicated its absence. First, when pterin-6-aldehyde or 6-hydroxymethylpterin was supplied to Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), or tomato (Lycopersicon esculentum) tissues, no reduction of the pterin ring was seen after 15 h, although reduction and oxidation of the side chain of pterin-6-aldehyde were readily detected. Second, no label was incorporated into folates when 6-[(3)H]hydroxymethylpterin was fed to cultured Arabidopsis plantlets for 7 d, whereas [(3)H]folate synthesis from p-[(3)H]aminobenzoate was extensive. Third, no NAD(P)H-dependent pterin ring reduction was found in tissue extracts. Genetic evidence showed a similar situation in Escherichia coli: a GTP cyclohydrolase I (folE) mutant, deficient in pterin synthesis, was rescued by dihydropterins but not by the corresponding oxidized forms. Expression of a trypanosomatid pterin reductase (PTR1) enabled rescue of the mutant by oxidized pterins, establishing that E. coli can take up oxidized pterins but cannot reduce them. Similarly, a GTP cyclohydrolase I (fol2) mutant of yeast (Saccharomyces cerevisiae) was rescued by dihydropterins but not by most oxidized pterins, 6-hydroxymethylpterin being an exception. These results show that the capacity to reduce oxidized pterins is not ubiquitous in folate-synthesizing organisms. If it is lacking, folate precursors or breakdown products that become oxidized will permanently exit the metabolically active pterin pool.  相似文献   

14.
The structure of the major fluorescent pterin present in thermophilic archaebacterium Sulfolobus solfataricus has been assigned, by analysis of the intact molecule and its hydrolytic and periodate cleavage products, as erythro-neopterin-3'-D-2-deoxy-2-aminoglucopyranoside. The trivial name solfapterin is proposed for this compound.  相似文献   

15.
The pathway of CO2 reduction to methane in Methanogenium tationis and Methanogenium thermophilicum is similar to that observed in other methanogens. In M. tationis a novel pterin, tatiopterin, is present. This pterin appears to be a structural and functional analog of methanopterin and sarcinapterin. Folate could not substitute for tatiopterin.  相似文献   

16.
B Wang  M S Jorns 《Biochemistry》1989,28(3):1148-1152
DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.  相似文献   

17.
Derivatives of methanopterin, a coenzyme involved in methanogenesis   总被引:16,自引:0,他引:16  
Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'-1' )O-alpha-ribofuranosyl-5'-phosphoric acid] aniline in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5'----1' glycosidic bond and yielded a 'hydrolytic product' which was identified as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7-methylpterin-6-carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6-ethyl-7-methyl-7,8-dihydropterin, 6-ethyl-7-methylpterin and a third compound, named methaniline which was identified as 4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'----1')O-alpha -ribofuranosyl-5'-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a L-glutamyl derivative of methanopterin, where the glutamate moiety is attached to the alpha-carboxylic acid group of the alpha-hydroxyglutaric acid moiety of methanopterin via an amide linkage.  相似文献   

18.
All azo colorants whose metabolism can liberate a carcinogenic arylamine, are suspected of having carcinogenic potential. Therefore, a new azo compound 4-phenethyl-5-[4-(1-(2-hydroxyethyl)-3,5-dimethyl-4-pyrazolylazo)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (substrate) was prepared to investigate its in vitro and in vivo biotransformation in rats by HPLC. Chromatographic separation of substrate and its metabolites was performed using a Chromasil C(18) column. The mobile phase consisted of acetonitrile and water in a linear gradient system. From the biotransformation of this compound, the reduction metabolite 4-(2-phenethyl)-5-(4-aminophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione was identified by comparing it to reference standard by HPLC-DAD. In the in vivo study, identification of the unknown peak which was the N-acetylation metabolite was confirmed by LC-MS spectrometry. Besides this, the azo compound was reduced to its corresponding amine in intestinal and cytosolic parts. In addition, oxidation of the methyl group and the phenyl ring, and reduction of azo group to hydrazo were identified in the cytosolic part using LC-MS.  相似文献   

19.
Dai P  Wang Y  Ye R  Chen L  Huang L 《Journal of bacteriology》2003,185(18):5500-5507
We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was capable of relaxing negatively supercoiled DNA at 75 degrees C in the presence of Mg2+. Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein. The S. solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion. The cleavage occurred only in the presence of a divalent cation, preferably Mg2+. The cofactor requirement of the enzyme was partially satisfied by Cu2+, Co2+, Mn2+, Ca2+, or Ni2+. It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg2+ and Ca2+). The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site. Analysis of cleavage by the S. solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5'-G(A/T)CA(T)AG(T)G(A)X / XX-3'. This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I. Based on these data and sequence analysis, we designate the enzyme S. solfataricus topoisomerase III.  相似文献   

20.
Robert H. White 《Chirality》1996,8(4):332-340
The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N5-N10-methenyl-5,6,7,8-tetrahydromethanopterin (N5-N10-methenyl-H4MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H4MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H4folate) show that the steps involved in the biological reduction of MPT to H4MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H4folate. © 1996 Wiley-Liss, Inc.  相似文献   

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