RESUMEN
The extracellular matrix is known to impact cell function during regeneration by modulating growth factor signaling. However, how the mechanical properties and structure of biomaterials can be used to optimize the cellular response to growth factors is widely neglected. Here, we engineered a macroporous biomaterial to study cellular signaling in environments that mimic the mechanical stiffness but also the mechanical heterogeneity of native extracellular matrix. We found that the mechanical interaction of cells with the heterogeneous and non-linear deformation properties of soft matrices (E < 5 kPa) enhances BMP-2 growth factor signaling with high relevance for tissue regeneration. In contrast, this effect is absent in homogeneous hydrogels that are often used to study cell responses to mechanical cues. Live cell imaging and in silico finite element modeling further revealed that a subpopulation of highly active, fast migrating cells is responsible for most of the material deformation, while a second, less active population experiences this deformation as an extrinsic mechanical stimulation. At an overall low cell density, the active cell population dominates the process, suggesting that it plays a particularly important role in early tissue healing scenarios where cells invade tissue defects or implanted biomaterials. Taken together, our findings demonstrate that the mechanical heterogeneity of the natural extracellular matrix environment plays an important role in triggering regeneration by endogenously acting growth factors. This suggests the inclusion of such mechanical complexity as a design parameter in future biomaterials, in addition to established parameters such as mechanical stiffness and stress relaxation.
Asunto(s)
Materiales Biocompatibles , Proteína Morfogenética Ósea 2 , Matriz Extracelular , Hidrogeles , Transducción de Señal , Proteína Morfogenética Ósea 2/metabolismo , Materiales Biocompatibles/química , Humanos , Matriz Extracelular/metabolismo , Hidrogeles/química , Animales , Ratones , Movimiento CelularRESUMEN
Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two ß-strands.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano , Proteínas Bacterianas , Glicósido Hidrolasas , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Amilosa/química , Amilosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Glucógeno/química , Glucógeno/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Glicosilación , Modelos Moleculares , Conformación ProteicaRESUMEN
Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 â 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 â 6)/(α1 â 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.
Asunto(s)
Geobacillus , Sistema de la Enzima Desramificadora del Glucógeno , Pan , Geobacillus/genética , Glucanos , Sistema de la Enzima Desramificadora del Glucógeno/genéticaRESUMEN
Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Bacterias/enzimología , Gelatina/química , Glucano 1,4-alfa-Glucosidasa/metabolismo , Glucógeno/metabolismo , alfa-Amilasas Pancreáticas/metabolismo , Almidón/química , Digestión , Hidrólisis , CinéticaRESUMEN
Glycogen is a highly branched α-glucan polymer widely used as energy and carbon reserve by many microorganisms. The branches are introduced by glycogen branching enzymes (EC 2.4.1.18), that are classified into glycoside hydrolase families 13 (GH13) and 57 (GH57). Most microorganisms have typically only a single glycogen branching enzyme (gbe) gene. Only a few microorganisms carry both GH13 and GH57 gbe genes, such as Petrotoga mobilis and Mycobacterium tuberculosis. Here we report the basic characteristics of the GH13 and GH57 GBE of P. mobilis, both heterologously expressed in E. coli. The GH13 GBE has a considerably higher branching activity towards the linear α-glucan amylose, and produces a highly branched α-glucan with a high molecular weight which is very similar to glycogen. The GH57 GBE, on the contrary, makes a much smaller branched α-glucan. While the GH13 GBE acts as a classical glycogen branching enzyme involved in glycogen synthesis, the role of GH57 GBE remains unclear.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Bacterias/enzimología , Glucógeno/biosíntesis , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Bacterias/genética , Activación Enzimática , Concentración de Iones de Hidrógeno , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Sistemas de Lectura Abierta , Especificidad por Sustrato , TemperaturaRESUMEN
AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary structural comparison suggest that AmyC could actually be a glycogen-branching enzyme (GBE), a key enzyme in the biosynthesis of glycogen. This communication presents phylogenetic and biochemical evidence that AmyC is a GBE with a relatively high hydrolytic (α-amylase) activity (up to 30% of the total activity), creating a branched α-glucan with 8.5% α-1,6-glycosidic bonds. The high hydrolytic activity is explained by the fact that AmyC has a considerably shorter catalytic loop (residues 213-220) not reaching the acceptor side. Secondly, in AmyC, the tryptophan residue (W 246) near the active site has its side chain buried in the protein interior, while the side chain is at the surface in Tk1436 and Tt1467 GBEs. The putative GBEs from three other Thermotogaceae, with very high sequence similarities to AmyC, were found to have the same structural elements as AmyC, suggesting that GH57 GBEs with relatively high hydrolytic activity may be widespread in nature.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Thermotoga maritima/enzimología , alfa-Amilasas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Hidrólisis , Modelos Moleculares , Filogenia , Conformación Proteica , Homología de Secuencia de Aminoácido , alfa-Amilasas/genéticaRESUMEN
Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/química , Glucanos/síntesis química , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Enzima Ramificadora de 1,4-alfa-Glucano/aislamiento & purificación , Amilopectina/química , Amilosa/química , Pruebas de Enzimas , Escherichia coli/genética , Hidrólisis , Peso Molecular , Thermus thermophilus/enzimologíaRESUMEN
SCOPE: Resistant starches (RSs) are classically considered to elicit health benefits through fermentation. However, it is recently shown that RSs can also support health by direct immune interactions. Therefore, it has been hypothesized that the structural traits of RSs might impact the health benefits associated with their consumption. METHODS AND RESULTS: Effects of crystallinity, molecular weight, and chain length distribution of RSs are determined on immune Toll-like receptors (TLRs), dendritic cells (DCs), and T-cell cytokines production. To this end, four type-3 RSs (RS3) are compared, namely Paselli WFR, JD150, debranched Etenia, and Amylose fraction V, which are extracted from potatoes and enzymatically modified. Dextrose equivalent seems to be the most important feature influencing immune signaling via activation of TLRs. TLR2 and TLR4 are most strongly stimulated. Especially Paselli WFR is a potent activator of multiple receptors. Moreover, the presence of amylose, even to residual levels, enhances DC and T-cell cytokine responses. Paselli WFR and Amylose fraction V influence T-cell polarization. CONCLUSIONS: It has been shown here that chain length and particularly dextrose equivalent are critical features for immune activation. This knowledge might lead to tailoring and design of immune-active RS formulations.
Asunto(s)
Citocinas/biosíntesis , Células Dendríticas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Almidón/farmacología , Receptores Toll-Like/fisiología , Amilosa/farmacología , Células Dendríticas/inmunología , Glucosa/farmacología , Humanos , Peso Molecular , Transducción de Señal/fisiología , Almidón/química , Células THP-1RESUMEN
4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1â4 linkages but mostly (relatively long) linear chains with α1â6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Limosilactobacillus reuteri/enzimología , Almidón/biosíntesisRESUMEN
4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-ß-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.
Asunto(s)
Archaeoglobus fulgidus/enzimología , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Glicósidos/metabolismo , Archaeoglobus fulgidus/genética , Biotransformación , Estabilidad de Enzimas , Glucósidos/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/química , Sistema de la Enzima Desramificadora del Glucógeno/genética , Calor , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Almidón/metabolismoRESUMEN
Dietary fibers are at the forefront of nutritional research because they positively contribute to human health. Much of our processed foods contain, however, only small quantities of dietary fiber, because their addition often negatively affects the taste, texture, and mouth feel. There is thus an urge for novel types of dietary fibers that do not cause unwanted sensory effects when applied as ingredient, while still positively contributing to the health of consumers. Here, we report the generation and characterization of a novel type of soluble dietary fiber with prebiotic properties, derived from starch via enzymatic modification, yielding isomalto/malto-polysaccharides (IMMPs), which consist of linear (α1 â 6)-glucan chains attached to the nonreducing ends of starch fragments. The applied Lactobacillus reuteri 121 GTFB 4,6-α-glucanotransferase enzyme synthesizes these molecules by transferring the nonreducing glucose moiety of an (α1 â 4)-glucan chain to the nonreducing end of another (α1 â 4)-α-glucan chain, forming an (α1 â 6)-glycosidic linkage. Once elongated in this way, the molecule becomes a better acceptor substrate and is then further elongated with (α1 â 6)-linked glucose residues in a linear way. Comparison of 30 starches, maltodextrins, and α-glucans of various botanical sources, demonstrated that substrates with long and linear (α1 â 4)-glucan chains deliver products with the highest percentage of (α1 â 6) linkages, up to 92%. In vitro experiments, serving as model of the digestive power of the gastrointestinal tract, revealed that the IMMPs, or more precisely the IMMP fraction rich in (α1 â 6) linkages, will largely pass the small intestine undigested and therefore end up in the large intestine. IMMPs are a novel type of dietary fiber that may have health promoting activity.
Asunto(s)
Proteínas Bacterianas/química , Fibras de la Dieta/análisis , Sistema de la Enzima Desramificadora del Glucógeno/química , Limosilactobacillus reuteri/enzimología , Polisacáridos/química , Prebióticos/análisis , Almidón/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Fibras de la Dieta/metabolismo , Digestión , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Humanos , Modelos Biológicos , Polisacáridos/metabolismo , Almidón/metabolismoRESUMEN
The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 â 4, 1 â 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 â 4) and (α1 â 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 â 4)/(α1 â 6) alternating structure.
Asunto(s)
Glicosiltransferasas/metabolismo , Limosilactobacillus reuteri/enzimología , Oligosacáridos/metabolismo , Sacarosa/metabolismoRESUMEN
Starch is an agricultural raw material used in many food and industrial products. It is present in granules that vary in shape in the form of amylose and amylopectin. Starch-degrading enzymes are used on a large scale in the production of sweeteners (high fructose corn syrup) and concentrated glucose syrups as substrate for the fermentative production of bioethanol and basic chemicals. Over the last two decades α-glucanotransferases (EC 2.4.1.xx), such as branching enzyme (EC 2.4.1.18) and 4-α-glucanotransferase (EC 2.4.1.25), have received considerable attention. These enzymes do not hydrolyze the starch as amylases do. Instead, α-glucanotransferases remodel parts of the amylose and amylopectin molecules by cleaving and reforming α-1,4- and α-1,6-glycosidic bond. Here we review the properties of α-glucanotransferases and discuss the emerging use of these enzymes in the generation of novel starch derivatives.
Asunto(s)
Amilopectina/química , Amilosa/química , Bacterias/enzimología , Biocatálisis , Sistema de la Enzima Desramificadora del Glucógeno/química , Enzima Ramificadora de 1,4-alfa-Glucano/química , Proteínas Bacterianas/química , Industria de Alimentos/métodos , Glucosiltransferasas/química , Hidrólisis , Conformación Molecular , Peso Molecular , Factores de TiempoRESUMEN
Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-D-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1â4)-α-D-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1â4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1â6 glycosidic linkages (α1â4 to α1â6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1â6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.
Asunto(s)
Glucanos/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Limosilactobacillus reuteri/enzimología , Oligosacáridos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , ADN Bacteriano/química , ADN Bacteriano/genética , Limosilactobacillus reuteri/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Glucansucrases are extracellular enzymes that synthesize a wide variety of α-glucan polymers and oligosaccharides, such as dextran. These carbohydrates have found numerous applications in food and health industries, and can be used as pure compounds or even be produced in situ by generally regarded as safe (GRAS) lactic acid bacteria in food applications. Research in the recent years has resulted in big steps forward in the understanding and exploitation of the biocatalytic potential of glucansucrases. This paper provides an overview of glucansucrase enzymes, their recently elucidated crystal structures, their reaction and product specificity, and the structural analysis and applications of α-glucan polymers. Furthermore, we discuss key developments in the understanding of α-glucan polymer formation based on the recently elucidated three-dimensional structures of glucansucrase proteins. Finally we discuss the (potential) applications of α-glucans produced by lactic acid bacteria in food and health related industries.
Asunto(s)
Biotecnología/métodos , Glucanos/química , Glucanos/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Secuencia de Aminoácidos , Tecnología de Alimentos , Lactobacillales , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
The probiotic bacterium Lactobacillus reuteri 121 produces two fructosyltransferase enzymes, a levansucrase and an inulosucrase. Although these two fructosyltransferase enzymes share high sequence similarity, they differ significantly in the type and size distribution of fructooligosaccharide products synthesized from sucrose, and in their activity levels. In order to examine the contribution of specific amino acids to such differences, 15 single and four multiple inulosucrase mutants were designed that affected residues that are conserved in inulosucrase enzymes, but not in levansucrase enzymes. The effects of the mutations were interpreted using the 3D structures of Bacillus subtilis levansucrase (SacB) and Lactobacillus johnsonii inulosucrase (InuJ). The wild-type inulosucrase synthesizes mostly fructooligosaccharides up to a degree of polymerization of 15 and relatively low amounts of inulin polymer. In contrast, wild-type levansucrase produces mainly levan polymer and fructooligosaccharides with a degree of polymerization < 5. Although most of the inulosucrase mutants in this study behaved similarly to the wild-type enzyme, the mutation G416E, at the rim of the active site pocket in loop 415-423, increased the hydrolytic activity twofold, without significantly changing the transglycosylation activity. The septuple mutant GM4 (T413K, K415R, G416E, A425P, S442N, W486L, P516L), which included two residues from the above-mentioned loop 415-423, synthesized 1-kestose only, but at low efficiency. Mutation A538S, located behind the general acid/base, increased the enzyme activity two to threefold. Mutation N543S, located adjacent to the +1/+2 sub-site residue R544, resulted in synthesis of not such a wide variety of fructooligosaccharides than the wild-type enzyme. The present study demonstrates that the product specificity of inulosucrase is easily altered by protein engineering, obtaining inulosucrase variants with higher transglycosylation specificity, higher catalytic rates and different fructooligosaccharide size distributions, without changing the ß(2-1) linkage type in the product.
Asunto(s)
Hexosiltransferasas/metabolismo , Limosilactobacillus reuteri/enzimología , Proteínas Mutantes/metabolismo , Oligosacáridos/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Dominio Catalítico , Hexosiltransferasas/química , Hexosiltransferasas/genética , Hidrólisis , Limosilactobacillus reuteri/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1 â 4) also (α1 â 6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)-DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.
Asunto(s)
Proteínas Bacterianas/química , Glucosiltransferasas/química , Limosilactobacillus reuteri/enzimología , Maltosa/química , Oligosacáridos/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía por Intercambio Iónico , Glucanos/química , Glicosilación , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/aislamiento & purificación , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Alcoholes del Azúcar/química , Trisacáridos/químicaRESUMEN
Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1â4 and synthesizing α1â6 and α1â4 glycosidic linkages) and α1â6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1â6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (ß/α)8 barrel that use sucrose to synthesize α-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1â6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.
Asunto(s)
Evolución Molecular , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Limosilactobacillus reuteri/enzimología , Secuencia de Aminoácidos , Análisis por Conglomerados , Glucanos/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/química , Limosilactobacillus reuteri/genética , Espectroscopía de Resonancia Magnética , Filogenia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Fructansucrases (FSs) catalyze a transfructosylation reaction with sucrose as substrate to produce fructo-oligosaccharides and fructan polymers that contain either ß-2,1 glycosidic linkages (inulin) or ß-2,6 linkages (levan). Levan-synthesizing FSs (levansucrases) have been most extensively investigated, while detailed information on inulosucrases is limited. Importantly, the molecular basis of the different product specificities of levansucrases and inulosucrases is poorly understood. We have elucidated the three-dimensional structure of a truncated active bacterial GH68 inulosucrase, InuJ of Lactobacillus johnsonii NCC533 (residues 145-708), in its apo form, with a bound substrate (sucrose), and with a transfructosylation product. The sucrose binding pocket and the sucrose binding mode are virtually identical with those of GH68 levansucrases, confirming that both enzyme types use the same fully conserved structural framework for the binding and cleavage of the donor substrate sucrose in the active site. The binding mode of the first transfructosylation product 1-kestose (Fru-ß(2-1)-Fru-α(2-1)-Glc, where Fru=fructose and Glc=glucose) in subsites -1 to +2 shows for the first time how inulin-type fructo-oligosaccharide bind in GH68 FS and how an inulin-type linkage can be formed. Surprisingly, observed interactions with the sugar in subsites +1 and +2 are provided by residues that are also present in levansucrases. The binding mode of 1-kestose and the presence of a more distant sucrose binding site suggest that residues beyond the +2 subsite, in particular residues from the nonconserved 1B-1C loop, determine product linkage type specificity in GH68 FSs.
Asunto(s)
Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Lactobacillus/enzimología , Sitios de Unión , Calcio/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hexosiltransferasas/genética , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (ß/α)(7)-fold catalytic domain A with an inserted domain B between ß2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.