Exo Manner Research Articles

Structural analysis of the reducing‐end xylose‐releasing exo‐oligoxylanase Rex8A from Paenibacillus barcinonensis BP‐23 deciphers its molecular specificity

Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillusbarcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides. The structure of the enzyme has been solved by X-ray crystallography showing a (α/α)6 folding common to GH8 enzymes. Analysis of inactived Rex8A-E70A complexed with xylotetraose revealed the existence of at least four binding subsites in Rex8A, with the oligosaccharide occupying subsites -3 to +1. The enzyme shows an extended Leu320-His321-Pro322 loop, common to other Rex, which blocks the binding of longer substrates to positive subsites further than +1 and seems responsible for the lack or diminished activity of Rex enzymes on xylan. Mutants with smaller residues in this loop failed to increase Rex8A activity on the polymer. Analysis of the complexes with AXOS showed the accommodation of arabinose at subsite -2, which cannot be allocated at subsite -1. Arabinose substitutions at the xylose O2 or O3 are accommodated by hydrophobic interaction and seem tolerated rather than recognized by Rex8A. A strained binding of the branch is facilitated by the lack of direct polar interactions of the xylose occupying this subsite, its water-mediated links allowing some conformational flexibility of the sugar. The plasticity of Rex8A is a notable property of the enzyme for its application in xylan deconstruction and upgrading. DATABASE: Structural data are available in PDB database under the accession numbers 6SRD (native form), 6TPP (E70A mutant in complex with EDO), 6TOW (E70A in complex with Xyl4), 6SUD (L320A mutant in complex with xylose), 6SHY (L320A/H321S double mutant in complex with EDO), 6TO0 (E70A in complex with AX3), and 6TRH (E70A in complex with AX4).

Access to functionalized pyrrolidones via copper-catalyzed aminooxygenation of unactivated alkenes

A simple and efficient copper-catalyzed aminooxygenation of unactivated alkenes with commercially available (2,2,6,6-tetramethylpiperidin-1-yl)-oxyl (TEMPO) as an oxygen source is reported. With this protocol, a variety of functionalized pyrrolidones were obtained in good to excellent yields, and good tolerance of functional groups and broad substrate scope were observed. Notably, the N-cyclization step selectively proceeded in an exo manner, and this protocol provides an efficient and straightforward approach to prepare vicinal amino alcohols.

Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity

The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose > cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-D-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a D-glucose concentration of 1000 mM, enzyme activity was 6.7-fold higher than that observed in the absence of D-glucose. With 31.3 mM D-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from a compost microbial metagenome may be a potent candidate for industrial applications.

Fungal Exo-acting Enzymes with Novel Catalytic Properties

Abstract: This article deals with characterizations of two Aspergillus niger exo-polygalacturonases (exo-PGs; EC 3.2.1.67) and a Penicillium chrysogenum exo-arabinanase (no EC number). Two exo-PGs, termed exo-PG1 and -PG2, purified from a commercial A. niger enzyme preparation (Pectinex AR) had molar masses of 82 and 56 kDa, respectively. Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Exo-PG1 had a broad specificity towards oligogalacturonates with different DPs, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid (GalA) and Xyl-GalA disaccharide, as identified by electrospray ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing GalA and acetylated GalA, as shown by ESI-ITMS. An exo-arabinanase, termed Abnx, was purified from a culture filtrate of P. chrysogenum 31B. The enzyme released only arabinobiose from the non-reducing terminus of α-1,5-L-arabinan and showed no activity towards p-nitrophenyl α-L-arabinofuranoside or α-1,5-L-arabinofuranobiose. The nucleotide sequence of the abnx cDNA gene, which encodes Abnx, was determined. Abnx was found to be structurally distinct from known arabinan-degrading enzymes based on its amino acid sequence and a hydrophobic cluster analysis (HCA). The abnx cDNA gene product expressed in Escherichia coli catalyzed the release of arabinobiose from α-1,5-L-arabinan. The activity of the recombinant Abnx towards a series of arabino-oligosaccharides, as expressed by kcat/Km value, was greatest with arabinohexaose. The recombinant enzyme was found to possess trans-arabinobiosylation activity on various acceptors, such as aliphatic alcohols, sugars and sugar alcohols. The transfer product of glycerol was identified as O-α-L-arabinosyl-(1→5)-O-α-L-arabinosyl-(1→1)-glycerol on the basis of the spectral data, fast atom bombardment-mass (FAB-MS) and 1H- and 13C-NMR. Unlike endo-arabinanases, Abnx catalyzed the hydrolysis of linear arabinan without inverting the anomeric configuration.

An exo β-1,3-glucanase synthesized de novo degrades lentinan during storage of Lentinule edodes and diminishes immunomodulating activity of the mushroom

Two β-1,3-glucanase isoenzymes, GNase I and II, were isolated from Lentinule edodes fruiting body. They degraded lentinan in an exo manner during storage of the mushroom. GNase I and II were monomeric proteins with apparent molecular masses of 72.9 and 74.7 kDa, respectively. It was shown that K m values for GNase I and GNase II against lentinan were 1.02 and 2.41 μM, respectively. Moreover, it was demonstrated by western blotting analysis that this enzyme was synthesized de novo in L. edodes during storage at 20 °C, but not at 1 or 5 °C. And this pattern of western blot was consistent with that of glucanase activity during storage of L. edodes. In addition, the immunomodulating effects of lentinan on TNF-α and NO production were diminished with its degradation by this enzyme. These results suggested that synthesis of exo β-1,3-glucanases were induced under storage conditions at 20 °C, and lentinan degradation by their attack might result in the depletion of immunomodulating activity of L. edodes.

Hydrolysis of mutan and prevention of its formation in streptococcal films by fungal α- d-glucanases

A mixed-linkage (α-1,3, α-1,6) water-insoluble glucan (known as mutan) of streptococcal origin was hydrolysed with fungal mutanase, dextranase and a mixture of these enzymes. The basic hydrolysis parameters, i.e. pH, temperature, reaction time, and enzyme and substrate concentration, were standardised to maximise sugar yield from glucan. Thus, the highest conversion of 0.02% mutan to soluble carbohydrate fraction (88% in 36 h) was obtained when mutanase and dextranase were combined. Under such conditions, the synergistic effects of these enzymes were seen as a considerable increase in hydrolysis yield, as compared to that obtained with mutanase (62%) or dextranase (18%) alone. Steady-state measurement of the products of the enzymic reaction during hydrolysis revealed that the mutanase operated in an exo manner and that the dextranase exhibited an endo type of action on mutan. HPLC analysis showed that glucose was the primary final product of glucan hydrolysis with mutanase. Digestion of biopolymer with dextranase or a mixture of mutanase and dextranase yielded glucose and its dimer as main hydrolytic products. Mutanase, dextranase and their mixture were also tested in various in vitro application studies. The enzymes were effective in preventing mutan formation and removing glucan from mixed streptococcal films, especially when the enzymic preparations were used in combination. However, these enzymes displayed less hydrolytic activity towards mutan, which had already been formed in the biofilm.

Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan

Two exo-polygalacturonases (EC 3.2.1.67) were purified from a commercial Aspergillus niger enzyme preparation by ammonium sulfate precipitation, preparative electrofocusing, anion-exchange and size-exclusion chromatographies. The enzymes had molar masses of 82 kDa (exo-PG1) and 56 kDa (exo-PG2). Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Addition of 0.01 mM HgCl 2 increased the exo-PG2 activity 3.4 times but did not affect exo-PG1. Analysis of the reaction products of (reduced) pentagalacturonate by high-performance anion-exchange chromatography revealed that both enzymes split the substrate from the non-reducing end in a multi-chain attack mode. Exo-PG1 had a broad specificity towards oligogalacturonates with different degrees of polymerisation, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid and Xyl–GalA disaccharide, as identified by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing galacturonic acid and acetylated galacturonic acid, as shown by ESI-ITMS.

Mechanism of asymmetric hydrogenation of alpha-(acylamino)acrylic esters catalyzed by BINAP-ruthenium(II) diacetate.

The mechanism of asymmetric hydrogenation of alpha-(acylamino)acrylic esters with Ru(CH(3)COO)(2)[(S)-binap] (BINAP = 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl), giving the S saturated products in >90% ee, has been investigated by means of a kinetic study, deuterium labeling experiments, isotope effect measurements, and NMR and X-ray analysis of certain Ru complexes. The hydrogenation in methanol under a low H2 pressure proceeds via a monohydride-unsaturate mechanism that involves the initial RuH formation followed by a reaction with an olefinic substrate. The migratory insertion in the enamide-RuH chelate complex occurs reversibly and endergonically in an exo manner, giving a five-membered metallacycle intermediate. The cleavage of the Ru-C bond is achieved with either H2 (major) or CH3OH (minor). Both of the pathways result in overall cis hydrogenation products. The hydrogen at C3 is mainly from an H2 molecule, and the C2 hydrogen is from another H2 or protic CH3OH. The major S and minor R enantiomers are produced via the same mechanism involving diastereomeric intermediates. The turnover rate is limited by the step of hydrogenolysis of a half-hydrogenated metallacyclic intermediate. The participation of two different hydrogen donor molecules is in contrast to the pairwise dihydrogenation using a single H2 molecule in the RhI-catalyzed reaction which occurs via a dihydride mechanism. In addition, the sense of asymmetric induction is opposite to that observed with S-BINAP-RhI catalysts. The origin of this phenomenon is interpreted in terms of stereocomplementary models of the enamide/metal chelate complexes. A series of model stoichiometric reactions mimicking the catalytic steps has indicated that most NMR-observable Ru complexes are not directly involved in the catalytic hydrogenation but are reservoirs of real catalytic complexes or even side products that retard the reaction.

Chemoenzymatic Synthesis of Glycopolypeptides Carrying α-Neu5Ac-(2 → 3)-β-[formula omitted]-Gal-(1 → 3)-α-[formula omitted]-GalNAc, β-[formula omitted]-Gal-(1 → 3)-α-[formula omitted]-GalNAc, and Related Compounds and Analysis of Their Specific Interactions with Lectins

Glycopolypeptide (1) carrying the β-d-Gal-(1 → 3)-α-d-GalNAc unit as a kind model of asialo-type mucin was synthesized through three steps: enzymatic synthesis of p-nitrophenyl disaccharide glycoside, reduction of the p-nitrophenyl group, and coupling of the amino group with the carboxyl group of poly(l-glutamic acid)s (PGA). In a similar manner, glycopolypeptides (2–7) carrying β-d-Gal-(1 → 3)-β-d-GalNAc, β-d-Gal-(1 → 3)-β-d-GlcNAc, β-d-Gal-(1 → 6)-α-d-GalNAc, β-d-Gal-(1 → 6)-β-d-GalNAc, α-d-GalNAc, and β-d-GalNAc, respectively, were synthesized as analogous polymers of polymer 1. Glycopolypeptides 8 and 9 as a mimic of sialo-type mucin were further prepared from polymers 1 and 2 as the acceptor of CMP-Neu5Ac by α2,3-(O)-sialyltransferase, respectively. Interactions of these glycopolypeptides with lectins were investigated with the double-diffusion test and the hemagglutination-inhibition assay and in terms of an optical biosensor based on surface plasmon resonance. Polymers 1 and 2 reacted strongly with peanut (Arachis hypogaea) agglutinin (PNA) and Agaricus bisporus agglutinin (ABA). On the other hand, polymers 8 and 9 through sialylation from polymers 1 and 2 reacted with ABA, but did not with PNA. Other polymers 3–7 did not show any reactivity for both the lectins. These results show that PNA acts precisely in an exo manner on the β-d-Gal-(1 → 3)-d-GalNAc sequence, while ABA acts in an endo manner. Polymers 6 and 7 substituted with GalNAc reacted strongly with soybean (Glycine max) agglutinin and Vicia villosa agglutinin B4, regardless of the configuration of the glycosidic linkage. The interaction of all polymers with Bauhinia purpurea agglutinin was much stronger than that of the corresponding sugars. Polymers 8 and 9 reacted with wheat germ (Triticum vulgaris) agglutinin (WGA), to which Neu5Ac residues are needed for binding, but polymers 1 and 2 did not. These sugar-substituted glycopolypeptides interacted specifically with the corresponding lectins. Furthermore, polymers 4–7 reacted with WGA, but the corresponding sugars did not. It suggests that the N-acetyl group along the PGA backbone has a cluster effect for WGA. The artificial glycopolypeptides were shown to be useful as tools and probes of carbohydrate recognition and modeling in the analysis of glycoprotein–lectin interactions.

A New Efficient Route to (±)-Physostigmine and (±)-Physovenine by Means of 5- exo Selective Aryl Radical Cyclization of o-Bromo- N-acryloylanilides

Bu 3SnH-mediated aryl radical cyclization of o-bromo- N-acryloylanilides 16 and 17 proceeded in a 5- exo manner exclusively to give, in high yields, oxindoles 3 and 4, the key intermediates for the synthesis of (±)-physostigmine and (±)-physovenine, respectively.

Preliminary characterization of a new exo-β-(1,4)-galactanase with transferase activity

As a prerequisite to the study of the fine chemical structure of the branched region of pectin, an exo-β-(1,4)-galactanase was purified from a commercial preparation (Pectinex AR). Purification was carried out by precipitation with 70% saturated ammonium sulfate, preparative electrofocusing, anion-exchange chromatography and affinity chromatography on cross-linked alginate. Exogalactanase specific activity was 992 nkat mg -1 and the enzyme was devoid of β-(1,3)- or β-(1,6)-galactanase, arabinanase, β- d-galactosidase and α- l-arabinofuranosidade activities. Residual exopolygalacturonase activity represented 2.9% of the galactanase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing showed two close bands with molecular weights of 120 000 and 90 000 and pHi of 3.8 and 4.1, respectively. The enzyme acted in an exo manner and its activity was optimum at pH 3.5 and 60°C. When incubated with galacto-oligosaccharides, new oligosaccharides with a higher degree of polymerization appeared, indicating the ability of the enzyme to transfer galactose residues.

Übergangsmetallkomplexe mit Schwefelliganden LIV. [Fe(CO) 2(′S 4′)] als synthon für den gezielten aufbau mehrkerniger heterometall-schwefel-zentren mit CO-bindungsstellen. Synthese, eigenschaften und röntgenstrukturanalyse von [Fe(CO) 2(μ-′S 4′){M(CO) 5} n] ( n = 1: M = Cr, Mo, W; n = 2: M = W; ′S 4′ 2− = 2,3,8,9-Dibenzo-1,4,7,10-tetrathiadecan(2 - ))

Diastereoselective Lewis acid base reactions between [Fe(CO) 2(′S 4′)] ( 1) and [M(CO) 5THF] (M = Cr, Mo, W) yield bi- and trinuclear heterometallic complexes [Fe(CO) 2(μ-′S 4′)i{M(CO) 5}] (M = Cr, 2a; Mo, 2b; W, 2c) and [Fe(CO) 2(μ-′S 4′) {W(CO) 5} 2] ( 3) respectively. 2a also is formed by the reaction of Li 2[μ-′S 4′{Cr(CO) 4} 2] with FeCl 2·4H 2O. X-Ray structure analyses of 2a and 3 show the M(CO) 5 fragments to coordinate the thiolato-S atoms of [Fe(CO) 2(′S 4′)] in an exo manner. The NMR spectra reveal this configuration is preserved in solution.

The interaction of nitrogen and sulphur donor macrocyclic ligands with dirhodium(II) tetracarboxylates

Reaction of dirhodium(II) tetracarboxylates [Rh 2(O 2CR) 4] with 1,4,8,11-tetra-azacyclotetradecane cyclam, L 1 under ambient conditions leads to the formation of the stable adducts [Rh 2O 2CR 4(cyclam)] n. Under similar conditions reaction of dirhodium(II) tetracarboxylates with the tridentate macrocycles 1,4,7-triazacyclononane (L 3), N, N′, N″- trimethyl-1,4,7-triazacyclononane (L 4) and 1,4,7-trithiacyclononane (L 5) gives adducts of 3:2 stoichiometry [{Rh 2(O 2CR) 4} 3(L) 2] n . The stoichiometries of these polymeric adducts illustrate the exo manner in which the macrocyclic ligands bind to the binuclear metal substrates.

Properties of Alginate Lyases from Marine Bacteria

Alginate lyases (EC 4.2.2.3) from two marine bacteria were isolated and partially characterized. A cell-bound lyase from isolate A3 had a molecular weight of approximately 100,000 and cleaved mannuronate blocks, apparently in an exo manner. A lyase recovered from the culture medium of isolate W3 was soluble in saturated ammonium sulfate, cleaved guluronate blocks, apparently in an endo manner, and had a molecular weight of 35,000. The thiobarbiturate test and urea-polyacrylamide gel electrophoresis were used to determine substrate specificity and mode of substrate cleavage by the enzymes.

Host-Pathogen Interactions

The fact that fungal glucans will stimulate soybeans to accumulate phytoalexins prompted an investigation of soybean cell beta-1,3-glucanases and beta-glucosidases, as well as the ability of these enzymes to hydrolyze the fungal glucans. Several beta-1,3-glucanases and beta-glucosidases can be solubilized from the walls of suspension-cultured soybean cells by treatment with 1.0 molar sodium acetate buffer. An enzyme, which has been termed beta-glucosylase I, is the dominant beta-1,3-glucanase in the cell wall extracts. Utilizing CM-Sephadex chromatography, hydroxylapatite chromatography, and affinity chromatography, beta-glucosylase I has been purified 71-fold, with 39% recovery, from the mixture of cell wall enzymes. The affinity chromatography column material was prepared by covalently attaching p-aminophenyl-1-beta-d-glucopyranoside, an analog of a beta-glucosylase I substrate, to Sepharose. beta-Glucosylase I, purified by this procedure, yields a single band on isoelectric focusing gels (pH 8.9). However, the purified beta-glucosylase I yields a darkly-staining protein band at an apparent molecular weight of 69,000 and several lightly-staining protein bands in sodium dodecyl sulfate polyacrylamide gels. Additional purification procedures fail to remove these lightly-staining protein bands.beta-Glucosylase I will hydrolyze the beta-glucan substrates, laminarin (3-linked) and lichenan (3- and 4-linked), and therefore, possesses beta-glucanase activity. Studies of the progressive hydrolysis of laminarin by beta-glucosylase I demonstrate that the enzyme hydrolyzes polysaccharide substrates in an exo manner. beta-Glucosylase I will also hydrolyze a variety of low molecular weight beta-glucosides including various beta-linked diglucosides. Thus, beta-glucosylase I also possesses beta-glucosidase activity.Several lines of evidence are presented that the beta-glucanase and the beta-glucosidase activities exhibited by purified beta-glucosylase I preparations are catalyzed by the same enzyme. This evidence includes inhibition studies which indicate that the beta-glucanase and the beta-glucosidase activities of beta-glucosylase I are catalyzed at the same active site. beta-Glucosylase I will also catalyze glucosyl transfer. This catalytic activity is responsible for the observed ability of the enzyme to synthesize di- and trisaccharides from laminarin. The disaccharides formed by beta-glucosylase I-catalyzed transglucosylation are the beta-anomers of the 6-, 4-, 3-, and 2-linked diglucosides in the relative proportions of 10:1:1:1. The ability of beta-glucosylase I to catalyze glucosyl transfer indicates that beta-glucosylase I is biochemically more similar to previously studied beta-glucosidases than to beta-glucanases. This conclusion is supported by the observation that beta-glucosylase I is strongly inhibited by 1,5-d-gluconolactone, an inhibitor of beta-glucosidases but not of beta-glucanases.

Furfurylamine誘導体と無水マレイン酸のDiels-Alder反応および立体化学

Diels-Alder reaction of furfurylamine derivatives (4a-g) with maleic anhydride is examined. Cycloaddition occurred highly stereoselective manner to give the lactam carboxylic acid (13a-g) in good to excellect yield. Evidences from chemical conversions and spectral examination revealed that the cycloaddition proceeded exclusively in exo manner in contrast to the ordinary Diels-Alder reaction.