Differential chain-length specificities of two isoamylase-type starch-debranching enzymes from developing seeds of kidney bean

Plant isoamylase-type starch-debranching enzymes (ISAs) hydrolyze alpha-1,6-linkages in alpha-1,4/alpha-1,6-linked polyglucans. Two ISAs, designated PvISA1/2 and PvISA3, were purified from developing seeds of kidney bean by ammonium sulfate fractionation and several column chromatographic procedures. The enzymes displayed different substrate specificities for polyglucans: PvISA1/2 showed broad chain-length specificities, whereas PvISA3 liberated specific chains with a DP of 2 to 4.     

胚乳中淀粉的合成

禾本科植物胚乳内所含有的淀粉根据其结构、组成可以分为两类：直链淀粉（由α-1,4糖苷键连接的多聚D-葡萄糖）和支链淀粉（在以α-1,4糖苷键连接的主链上通过形成α-1,6糖苷键引入支链的多聚D-葡萄糖）。前者是以一种线性无序状态存在,而支链淀粉则是构成淀粉半晶体结构的主要成分。其中,除了负责合成作为糖基直接供体的ADP—Glc的酶AGPase外,直链淀粉中链的延伸反应由GBSSI完成,而支链淀粉的合成则相对复杂,需要SS、SBE、DBE、SP等一些酶的协同调控来共同完成。本文综述了胚乳中淀粉合成过程中所涉及的一些关键酶的研究进展,并对此研究领域进行了展望。     

Screening and Application of Microbial Esterases for the Enantioselective Synthesis of Chiral Glycerol Derivatives

Glucansucrases from family 70 of glycoside-hydrolases catalyse the synthesis of α-glucans with various types of osidic linkages from sucrose. Among these enzymes, alternansucrase (ASR) and dextransucrase E (DSR-E) catalyse the formation of unusual α-glucans. ASR catalyses the synthesis of linear glucan with α-1,3 and α-1,6 alternating linkages and DSR-E synthesizes a glucan containing α-1,6 linkages in the linear chain and α-1,2 branches. The sequence analysis of these enzymes enabled the identification of structural elements suspected to be involved in the enzyme specificities. Biochemical characterization of ASR and DSR-E variants obtained from gene truncations or site-directed mutagenesis experiments showed that the specificity of these enzymes to form different types of osidic linkage is controlled by two different approaches. For ASR, the double specificity is controlled by only one catalytic domain where important amino acids involved in the enzyme specificity have been identified. In the case of DSR-E, the double specificity is controlled by two different catalytic domains both belonging to family 70, each domain being specific of one type of linkage.     

Action of alpha-1,6-glucan glucohydrolase on oligosaccharides derived from dextran

The action of α-1,6-glucan glucohydrolase on α-(1→6)-D-glucosidic linkages in oligosaccharides that also contain an α-(1→2)-, α-(1→3)-, or α-(1→4)-D-glucosidic linkage has been investigated. The enzyme could hydrolyse α-(1→6)-D-glucosidic linkages from the non-reducing end, including those adjacent to an anomalous linkage. α-(1→6)-D-Glucosidic linkages at branch points were not hydrolysed, and the enzyme could neither hydrolyse nor by-pass the anomalous linkages. These properties of α-1,6-glucan glucohydrolase explain the limited hydrolysis of dextrans by the exo-enzyme. Hydrolysis of the main chain of α-(1→6)-D-glucans will always stop one D-glucose residue away from a branch point. The extent of hydrolysis by α-1,6-glucan glucohydrolase of some oligosaccharide products of the action on dextran of Penicillium funiculosum and P. lilacinum dextranase, respectively, has been compared. Differences in the specificity of the two endo-dextranases were revealed. The Penicillium enzymes may hydrolyse dextran B-512 to produce branched oligosaccharides that retain the same 1-unit and 2-unit side-chains that occur in dextran.     

Strict order of (Fuc to Asn-linked GlcNAc) fucosyltransferases forming core-difucosylated structures

In insect cells fucose can be either α1,6- or α1,3-linked to the asparagine-bound GlcNAc residue of N-glycans. Difucosylated glycans have also been found. Kinetic studies and acceptor competition experiments demonstrate that two different enzymes are responsible for this α1,6- and α1,3-linkage of fucose. Using dansylated acceptor substrates a strict order of these enzymes can be established for the formation of difucosylated structures. First, the α1,6-fucosyltransferase catalyses the transfer of fucose into α1,6-linkage to the non-fucosylated acceptor and then the α1,3-fucosyltransferase completes the difucosylation. © 1998 Rapid Science Ltd     

Sequential processing of mannose-containing glycans by two α-mannosidases from <Emphasis Type="Italic">Solitalea canadensis</Emphasis>

Two putative α-mannosidase genes isolated from the rather unexplored soil bacterium Solitalea canadensis were cloned and biochemically characterised. Both recombinant enzymes were highly selective in releasing α-linked mannose but no other sugars. The α-mannosidases were designated Sca2/3Man2693 and Sca6Man4191, and showed the following biochemical properties: the temperature optimum for both enzymes was 37 °C, and their pH optima lay at 5.0 and 5.5, respectively. The activity of Sca2/3Man2693 was found to be dependent on Ca²⁺ ions, whereas Cu²⁺ and Zn²⁺ ions almost completely inhibited both α-mannosidases. Specificity screens with various substrates revealed that Sca2/3Man2693 could release both α1-2- and α1-3-linked mannose, whereas Sca6Man4191 only released α1-6-linked mannose. The combined enzymatic action of both recombinant α-mannosidases allowed the sequential degradation of high-mannose-type N-glycans. The facile expression and purification procedures in combination with strict substrate specificities make α-mannosidases from S. canadensis promising candidates for bioanalytical applications.     

De novo Synthesis of Polyglucans by a Phosphorylase Isoenzyme in Algae

The α2 phosphorylase isoenzyme of Oscillatoria princeps, Rhodymenia pertusa and Chlorella pyrcnoidosa has been found to be capable of the synthesis of amylose-like polyglucans without the necessity for addition of primers. This enzyme differs in this respect from the a1 isoenzyme of blue-green and green algae. The a2 enzyme appears to be a glyco-protein and synthesis of linear polyglucans appears to occur by the apposition of glucosyl residues from glucose-1-phos-phate directly to the glycosyl moiety of its molecule.     

Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the α-1,6-glucosidic linkage of isomaltooligosaccharides from their non-reducing ends to produce α-glucose. By using the mutant of catalytic acid Glu236→Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 Å resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (β/α)₈-barrel in domain A, which is common to the α-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites −1 to +2. The environment of the glucose residue at subsite −1 is similar to the environment of this residue in the α-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite −1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile α-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile α-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the α-amylase structure from Bacillus subtilis. The comparison with the α-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of α-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes.     

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of α1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of α1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K_d) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for α1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K_a) for α1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were α1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of α1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.     

Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme

Glycogen debranching enzyme (GDE) in mammals and yeast exhibits α-1,4-transferase and α-1,6-glucosidase activities within a single polypeptide chain and facilitates the breakdown of glycogen by a bi-functional mechanism. Each enzymatic activity of GDE is suggested to be associated with distinct domains; α-1,4-glycosyltransferase activity with the N-terminal domain and α-1,6-glucosidase activity with the C-terminal domain. Here, we present the biochemical features of the GDE from Saccharomyces cerevisiae using the substrate glucose(n)-β-cyclodextrin (Gn-β-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1–644) showed α-1,4-transferase activity on maltotetraose (G4) and G4-β-CD, yielding various lengths of (G)_n. Surprisingly, the N-terminal domain also exhibited α-1,6-glucosidase activity against G1-β-CD and G4-β-CD, producing G1 and β-CD. Mutational analysis showed that residues D535 and E564 in the N-terminal domain are essential for the transferase activity but not for the glucosidase activity. These results indicate that the N-terminal domain (1–644) alone has both α-1,4-transferase and the α-1,6-glucosidase activities and suggest that the bi-functional activity in the N-domain may occur via one active site, as observed in some archaeal debranching enzymes.     

Immobilization of Chloroplast Photosystems

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.     

Purification and characterization of highly branched α-glucan-producing enzymes from Paenibacillus sp. PP710

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.     

Specificity of Debranching Enzymes

THE debranching enzymes are a most important group because, with the amylases and phosphorylases, they completely degrade starch and glycogen to the monosaccharide level. Some of the earlier preparations of debranching enzymes were not homogeneous and their use led to erroneous conclusions about their specificity. These views were later qualified by studies using more highly purified preparations. For example, some preparations of yeast isoamylase hydrolysed α-1,6-D-glucosidic linkages in both glycogen and oligosaccharide α-dextrins¹, but the latter activity is now no longer ascribed to isoamylase².     

GH62 arabinofuranosidases: Structure,function and applications

Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. α-1,2 and α-1,3 arabinofuranosyl specific α-l-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release of α-l-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies α-l-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively α-l-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 α-l-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.     

Synthesis of 2-Deoxy-glucooligosaccharides through Condensation of 2-Deoxy-D-glucose by Glucoamylase and α-Glucosidase

Glucoamylases from Aspergillus niger and Rhizopus niveus catalyzed condensation of 2-deoxy-D-glucose (dGlc) to yield deoxy-glucooligosaccharides with polymerization degrees of 2–5. The enzymes also gave a small amount of products from 3-deoxy-o-glucose, but no products from 6-deoxy-D-glucose. A. niger α-glucosidase also catalyzed condensation of dGlc, while Torula and Saccharomyces α-glucosidases had low activity. α-l,4-, 1,6-, and 1,3-linked deoxy-glucobioses were isolated and identified as the products of A. niger glucoamylase and A. niger α-glucosidase. In the reaction of the glucoamylase, 1,4- and 1,3-linked saccharides decreased with an increase of 1,6-linked one. A. niger α-glucosidase produced α-1,6-linked disaccharide predominantly during the whole course of the reaction.     

Val216 decides the substrate specificity of alpha-glucosidase in Saccharomyces cerevisiae.

Differences in the substrate specificity of alpha-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzing alpha-1,4- and alpha-1,6-glucosidic linkages, two alpha-glucosidases, maltase and isomaltase, from Saccharomyces cerevisiae were cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues, respectively. There was 72.1% sequence identity with 165 amino acid alterations between the two alpha-glucosidases. These two alpha-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified alpha-glucosidases showed the same substrate specificities as those of their parent native glucosidases. Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities. When the consensus region II, which is one of the four regions conserved in family 13 (alpha-amylase family), is replaced with the maltase type, the chimeric enzymes alter to hydrolyze maltose. Three amino acid residues in consensus region II were different in the two alpha-glucosidases. Thus, we modified Val216, Gly217, and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis. The Val216 mutant was altered to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity, indicating that Val216 is an important residue discriminating the alpha-1,4- and 1,6-glucosidic linkages of substrates.     

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.     

Wound debridement potential of glycosidases of the wound-healing maggot, Lucilia sericata

The wound-healing maggot, Lucilia sericata Meigen (Diptera: Calliphoridae), degrades extracellular matrix components by releasing enzymes. The purpose of this study was to investigate the glycosylation profiles of wound slough/eschar from chronic venous leg ulcers and the complementary presence of glycosidase activities in first-instar excretions/secretions (ES1) and to define their specificities. The predominant carbohydrate moieties present in wound slough/eschar were determined by probing one-dimensional Western blots with conjugated lectins of known specificities. The presence of specific glycosidase activities in ES1 was determined using chromogenic and fluorogenic substrates. The removal of carbohydrate moieties from slough/eschar proteins by glycosidases in ES1 was determined by two-dimensional electrophoresis and Emerald 300 glycoprotein staining. α-d-glucosyl, α-d-mannosyl and N-acetlyglucosamine residues were detected on slough/eschar-derived proteins. Furthermore, it was demonstrated that the treatment of slough/eschar with ES1 significantly reduced uptake of the carbohydrate-specific stain. Subsequently, α-d-glucosidase, α-d-mannosidase and N-acetylglucosaminidase activities were identified in ES1. Specific chromogenic and fluorogenic substrates and gel filtration chromatography showed that these activities result from distinct enzymes. These activities were mirrored in the removal of α-d-glucosyl, α-d-mannosyl and N-acetylglucosamine residues from proteins of slough/eschar from maggot-treated wounds. These data suggest that maggot glycosidases remove sugars from slough/eschar proteins. This may contribute to debridement, which is ultimately accomplished by a suite of biochemically distinct enzymes present in ES1.