RESUMO
Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.
RESUMO
The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses ß-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming ß-1,3 glucan acceptor, making a ß-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several ß-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-ß-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-ß-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.
Assuntos
Flavobacteriaceae , Oligossacarídeos , Filogenia , Oligossacarídeos/química , Polissacarídeos , Especificidade por SubstratoRESUMO
Alginate (alginic acid) is a linear polysaccharide, wherein (1â4)-linked ß-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1â4) glycosidic linkages between the monomers by a ß-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at â¼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is â¼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.
RESUMO
Amphipathic copolymers such as poly(styrene-maleic acid) (SMA) are promising tools for the facile extraction of membrane proteins (MPs) into native nanodiscs. Here, we designed and synthesized a library of well-defined alternating copolymers of SMA analogues in order to elucidate polymer properties that are important for MP solubilization and stability. MP extraction efficiency was determined using KcsA from E. coli membranes, and general solubilization efficiency was investigated via turbidimetry experiments on membranes of E. coli, yeast mitochondria, and synthetic lipids. Remarkably, halogenation of SMA copolymers dramatically improved solubilization efficiency in all systems, while substituents on the copolymer backbone improved resistance to Ca2+. Relevant polymer properties were found to include hydrophobic balance, size and positioning of substituents, rigidity, and electronic effects. The library thus contributes to the rational design of copolymers for the study of MPs.
Assuntos
Proteínas de Membrana , Poliestirenos , Escherichia coli , Interações Hidrofóbicas e Hidrofílicas , Maleatos/química , Proteínas de Membrana/química , Polímeros , Poliestirenos/químicaRESUMO
Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(ß1â4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.
Assuntos
Galinhas , Infecções por Clostridium , Clostridium perfringens , Lipopolissacarídeos , Doenças das Aves Domésticas , Ácidos Teicoicos , Animais , Galinhas/imunologia , Galinhas/microbiologia , Infecções por Clostridium/imunologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/química , Clostridium perfringens/imunologia , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/prevenção & controle , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/farmacologiaRESUMO
Glycosaminoglycans (GAGs) were extracted from heads of silver-banded whiting (SBW) fish and subjected to preliminary biocompatibility testing per ISO 10993: intracutaneous irritation, maximization sensitization, systemic toxicity, and cytotoxicity. When the GAG solution was injected intradermally, the observed irritation was within ISO limits and comparable to a marketed control. There was no evidence of sensitization, systemic toxicity, or cellular toxicity on the test organisms treated with the GAG mixture from SBW fish heads. Fractionation by size-exclusion chromatography has shown three distinct fractions: F1 as low molecular weight hyaluronic acid (190 kDa), F2 (82 kDa) and F3 (64 kDa), both as chondroitin sulfates. Structural characterization by 1D and 2D nuclear magnetic resonance spectroscopy and disaccharide analysis have shown sulfation ratios at positions C4:C6 of the F2 and F3 fractions respectively as 70:20% and 50:30%, and the balance of non-sulfated and 4,6-di-sulfated units. The preliminary results here suggest that GAG-based extracts from SBW fish heads are suitable alternative products to be used in soft tissue augmentation, although further long-term biocompatibility studies are still required.
Assuntos
Materiais Biocompatíveis/química , Glicosaminoglicanos/química , Mariposas/química , Animais , Materiais Biocompatíveis/isolamento & purificação , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Fracionamento Químico , Cromatografia em Gel , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/farmacologia , Ácido Hialurônico/química , Camundongos , Estrutura Molecular , Análise EspectralRESUMO
Lipopolysaccharides (LPS) are among the known pathogen-associated molecular patterns (PAMPs). LPSs are potent elicitors of PAMP-triggered immunity (PTI), and bacteria have evolved intricate mechanisms to dampen PTI. Here we demonstrate that Xylella fastidiosa (Xf), a hemibiotrophic plant pathogenic bacterium, possesses a long chain O-antigen that enables it to delay initial plant recognition, thereby allowing it to effectively skirt initial elicitation of innate immunity and establish itself in the host. Lack of the O-antigen modifies plant perception of Xf and enables elicitation of hallmarks of PTI, such as ROS production specifically in the plant xylem tissue compartment, a tissue not traditionally considered a spatial location of PTI. To explore translational applications of our findings, we demonstrate that pre-treatment of plants with Xf LPS primes grapevine defenses to confer tolerance to Xf challenge.
Assuntos
Imunidade Inata/imunologia , Lipopolissacarídeos/imunologia , Antígenos O/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal/imunologia , Xylella/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Lipopolissacarídeos/metabolismo , Antígenos O/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Vitis/genética , Vitis/imunologia , Vitis/microbiologia , Xylella/metabolismo , Xylella/fisiologiaRESUMO
Exopolysaccharides produced by lactic acid bacteria are extensively used for food applications. Glucansucrase enzymes of lactic acid bacteria use sucrose to catalyze the synthesis of α-glucans with different linkage compositions, size and physico-chemical properties. Crystallographic studies of GTF180-ΔN show that at the acceptor binding sites +1 and +2, residue W1065 provides stacking interactions to the glucosyl moiety. However, the detailed functional roles of W1065 have not been elucidated. We performed random mutagenesis targeting residue W1065 of GTF180-ΔN, resulting in the generation of 10 mutant enzymes that were characterized regarding activity and product specificity. Characterization of mutant enzymes showed that residue W1065 is critical for the activity of GTF180-ΔN. Using sucrose, and sucrose (donor) plus maltose (acceptor) as substrates, the mutant enzymes synthesized polysaccharides and oligosaccharides with changed linkage composition. The stacking interaction of an aromatic residue at position 1065 is essential for polysaccharide synthesis.
Assuntos
Ligação Genética/genética , Glicosiltransferases/genética , Mutação/genética , Oligossacarídeos/genética , Polissacarídeos/genética , Glicosiltransferases/química , Limosilactobacillus reuteri/enzimologia , Limosilactobacillus reuteri/genética , Maltose/biossíntese , Maltose/química , Maltose/genética , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Polissacarídeos/biossíntese , Polissacarídeos/química , Estrutura Secundária de Proteína , Sacarose/química , Sacarose/metabolismoRESUMO
BACKGROUND: To reduce the cost of the enzymes for the hydrolysis of lignocellulosic biomass, two main strategies have been followed: one, the reduction of enzyme dosing by the use of more efficient and stable enzymatic cocktails; another, to include accessory enzymes in the cocktails to increase yields by reducing the recalcitrant carbohydrate fraction remaining at the end of the process. To guide this second strategy, we have explored the chemical bond composition of different fractions of recalcitrant carbohydrates after enzymatic hydrolysis. RESULTS: Two lignocellulosic feedstocks of relevance for the biofuels industry have been analyzed, corn stover and sugarcane straw. On comparing the composition of chemical bonds of the starting pretreated material with samples after standard and forced hydrolysis (with enzyme overdosing), we obtained similar sugar and chemical bond composition. CONCLUSIONS: This suggests that the current enzymatic cocktails bear the set of enzymes needed to hydrolyze these feedstocks. From our point of view, the results show the need for a parallel fine-tuning of the enzymatic cocktails with the pretreatment process to maximize sugar release yield.
RESUMO
The glucansucrase GTFA of Lactobacillus reuteri 121 produces an α-glucan (reuteran) with a large amount of alternating (α1 â 4) and (α1 â 6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) with GTFA of L. reuteri 121; it also synthesizes an α-glucan with (α1 â 4) and (α1 â 6) linkages, but with a clearly different ratio compared to GTFA. In the present study, we show that residues in loop977 (970DGKGYKGA977) and helix α4 (1083VSLKGA1088) are main determinants for the linkage specificity difference between GTFO and GTFA, and hence are important for the synthesis of alternating (α1 â 4) and (α1 â 6) linkages in GTFA. More remote acceptor substrate binding sites (i.e.+3) are also involved in the determination of alternating linkage synthesis, as shown by structural analysis of the oligosaccharides produced using panose and maltotriose as acceptor substrate. Our data show that the amino acid residues at acceptor substrate binding sites (+1, +2, +3 ) together form a distinct physicochemical micro-environment that determines the alternating (α1 â 4) and (α1 â 6) linkages synthesis in GTFA.
Assuntos
Glicosiltransferases/química , Limosilactobacillus reuteri/enzimologia , Oligossacarídeos/química , Proteínas de Bactérias/química , Sítios de Ligação , Glucanos/química , Hidrólise , Espectroscopia de Ressonância Magnética , Maltose/química , Mutagênese Sítio-Dirigida , Mutação , Polissacarídeos/química , Probióticos , Especificidade por Substrato , Sacarose/químicaRESUMO
By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.
Assuntos
Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Oxirredutases/metabolismo , Sordariales/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Dissacarídeos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos Moleculares , Oxirredução , Oxirredutases/química , Conformação Proteica , Alinhamento de Sequência , Sordariales/química , Sordariales/metabolismo , Especificidade por Substrato , Xilanos/metabolismoRESUMO
Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (ß1â3) linkages preferably and to a lesser extent (ß1â6) or (ß1â4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (ß1â3)(ß1â6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic ß-glucans in B. diazoefficiens.
Assuntos
Bradyrhizobium/enzimologia , Oligossacarídeos/metabolismo , beta-Glucosidase/metabolismo , Biocatálise , Proteínas Recombinantes/metabolismo , beta-Glucosidase/genéticaRESUMO
Lactic acid bacteria (LAB) produce exopolysaccharides (EPS) that are important for biofilm formation in the mammalian oral cavity and gastrointestinal tract. Sucrose is a well-known substrate for homo-EPS formation by Lactobacillus reuteri glucansucrases (GS). Starch is the main fermentable carbohydrate in the human diet, and often consumed simultaneously with sucrose. Recently we have characterized L. reuteri strains that also possess 4,6-α-glucanotransferases (4,6-α-GTases) that act on starch yielding isomalto-/malto-polysaccharides. In this study we have characterized the EPS formed by L. reuteri 35-5 cells and enzymes from sucrose plus starch. The data show that both in vivo and in vitro the L. reuteri 35-5 GS and 4,6-α-GTase enzymes, incubated with sucrose plus starch, cross-react and contribute to synthesis of the final hybrid EPS products. This may have strong effects on the EPS functional properties, influence biofilm formation, and affect the relationship between dietary intake of sucrose and starch, and dental caries formation.
Assuntos
Biopolímeros/biossíntese , Limosilactobacillus reuteri/metabolismo , Polissacarídeos/biossíntese , Amido/metabolismo , Sacarose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/genéticaRESUMO
The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.
Assuntos
Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Vesículas Transportadoras/metabolismo , Tularemia/imunologia , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Francisella tularensis/genética , Francisella tularensis/metabolismo , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Antígenos O/imunologia , Vesículas Transportadoras/genética , Tularemia/microbiologia , Tularemia/prevenção & controle , VacinaçãoRESUMO
α-Glucans produced by glucansucrase enzymes of lactic acid bacteria attract strong attention as novel ingredients and functional biopolymers in the food industry. In the present study, α-helix 4 amino acid residues D1085, R1088, and N1089 of glucansucrase GTF180 of Lactobacillus reuteri 180 were targeted for mutagenesis both jointly and separately. Analysis of the mutational effects on enzyme function revealed that all D1085 and R1088 mutants catalyzed the synthesis of hyperbranched α-glucans with 15-22% branching (α1â3,6) linkages, compared to 13% in the wild-type GTF180. In addition, besides native (α1â6) and (α1â3) linkages, all of the mutations introduced a small amount of (α1â4) linkages (5% at most) in the polysaccharides produced. We conclude that α-helix 4 residues, especially D1085 and R1088, constituting part of the +2 acceptor binding subsite, are important determinants for the linkage specificity. The new hyperbranched α-glucans provide very interesting structural diversities and may find applications in the food industry.
Assuntos
Proteínas de Bactérias/metabolismo , Glucanos/biossíntese , Glucanos/química , Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Sacarose/metabolismo , Proteínas de Bactérias/genética , Glicosiltransferases/genética , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/metabolismo , Estrutura Molecular , Mutagênese , MutaçãoRESUMO
α-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-ΔN from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite +1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (α1 â 6) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.
Assuntos
Aminoácidos/metabolismo , Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Sequência de Aminoácidos , Aminoácidos/química , Domínio Catalítico , Cromatografia Gasosa-Espectrometria de Massas , Glicosiltransferases/química , Glicosiltransferases/genética , Metilação , Dados de Sequência Molecular , Mutagênese , Espectroscopia de Prótons por Ressonância Magnética , Homologia de Sequência de AminoácidosRESUMO
GTFA, a glucansucrase enzyme of the probiotic bacterium Lactobacillus reuteri 121, is capable of synthesizing an α-glucan polysaccharide with (1 â 4) and (1 â 6) linkages from sucrose. With respect to its biosynthesis, the present study has shown that the ratio of oligosaccharide versus polysaccharide synthesized was directly proportional to the concentration of sucrose. It appears that the size distribution of products is kinetically controlled, but the linkage distribution in the polysaccharide material is not changed. At high sucrose concentrations the sucrose isomers leucrose and trehalulose were synthesized, using the accumulated fructose as acceptor, together with 4'- and 6'-α-D-glucosyl-leucrose and 6'-α-D-glucosyl-trehalulose. The finding of an additional branched hexasaccharide demonstrates that the enzyme is able to introduce branch-points already in relatively short oligosaccharides.
Assuntos
Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Polissacarídeos/metabolismo , Sacarose/metabolismo , Proteínas de Bactérias/metabolismo , Cinética , Limosilactobacillus reuteri/metabolismo , Oligossacarídeos/metabolismoRESUMO
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.
Assuntos
Archaeoglobus fulgidus/enzimologia , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicosídeos/metabolismo , Archaeoglobus fulgidus/genética , Biotransformação , Estabilidade Enzimática , Glucosídeos/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Amido/metabolismoRESUMO
Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide chain follows a U shape course to form five domains, including domains A, B, and C, which resemble those of family GH13 enzymes, and two extra and novel domains (domains IV and V), which are attached to the catalytic core. To elucidate the functional roles of domain V, we have deleted the domain V fragments from both the N- and C-terminal ends (GTF180-ΔNΔV). Truncation of domain V of GTF180-ΔN yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. Instead, GTF180-ΔNΔV produced a large amount of oligosaccharides. Domain V is not involved in determining the linkage specificity, and the size of polysaccharide produced as the polysaccharide produced by GTF180-ΔNΔV was identical in size and structure with that of GTF180-ΔN. The data indicates that GTF180-ΔNΔV acts nonprocessively, frequently initiating synthesis of a new oligosaccharide from sucrose, instead of continuing the synthesis of a full size polysaccharide. Mutations L940E and L940F in GTF180-ΔNΔV, which are involved in the acceptor substrate binding, restored polysaccharide synthesis almost to the level of GTF180-ΔN. These results demonstrated that interactions of growing glucan chains with both domain V and acceptor substrate binding sites are important for polysaccharide synthesis.
Assuntos
Glicosiltransferases/metabolismo , Limosilactobacillus reuteri/enzimologia , Limosilactobacillus reuteri/metabolismo , Polissacarídeos/biossíntese , Glicosiltransferases/genética , Limosilactobacillus reuteri/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Deleção de SequênciaRESUMO
The common saprophyte Aspergillus niger may experience carbon starvation in nature as well as during industrial fermentations. Starvation survival strategies, such as conidiation or the formation of exploratory hyphae, require energy and building blocks, which may be supplied by autolysis. Glycoside hydrolases are key effectors of autolytic degradation of fungal cell walls, but knowledge on their identity and functionality is still limited. We recently identified agnB and cfcA as two genes encoding carbohydrate-active enzymes that had notably increased transcription during carbon starvation in A. niger. Here, we report the biochemical and functional characterization of these enzymes. AgnB is an α-1,3-glucanase that releases glucose from α-1,3-glucan substrates with a minimum degree of polymerization of 4. CfcA is a chitinase that releases dimers from the nonreducing end of chitin. These enzymes thus attack polymers that are found in the fungal cell wall and may have a role in autolytic fungal cell wall degradation in A. niger. Indeed, cell wall degradation during carbon starvation was reduced in the double deletion mutant ΔcfcA ΔagnB compared to the wild-type strain. Furthermore, the cell walls of the carbon-starved mycelium of the mutant contained a higher fraction of chitin or chitosan. The function of at least one of these enzymes, CfcA, therefore appears to be in the recycling of cell wall carbohydrates under carbon limiting conditions. CfcA thus may be a candidate effector for on demand cell lysis, which could be employed in industrial processes for recovery of intracellular products.