RESUMO
Complete enzymatic degradation of plant polysaccharides is a result of combined action of various carbohydrate-active enzymes (CAZymes). In this paper, we demonstrate the potential of the filamentous fungus Scytalidium candidum 3C for processing of plant biomass. Structural annotation of the improved assembly of S. candidum 3C genome and functional annotation of CAZymes revealed putative gene sequences encoding such proteins. A total of 190 CAZyme-encoding genes were identified, including 104 glycoside hydrolases, 52 glycosyltransferases, 28 oxidative enzymes, and 6 carbohydrate esterases. In addition, 14 carbohydrate-binding modules were found. Glycoside hydrolases secreted during the growth of S. candidum 3C in three media were analyzed with a variety of substrates. Mass spectrometry analysis of the fungal culture liquid revealed the presence of peptides identical to 36 glycoside hydrolases, three proteins without known enzymatic function belonging to the same group of families, and 11 oxidative enzymes. The activity of endo-hemicellulases was determined using specially synthesized substrates in which the glycosidic bond between monosaccharide residues was replaced by a thio-linkage. During analysis of the CAZyme profile of S. candidum 3C, four ß-xylanases from the GH10 family and two ß-glucanases from the GH7 and GH55 families were detected, partially purified, and identified.
Assuntos
Ascomicetos , Proteínas Fúngicas , Genoma Fúngico , Oxirredutases , Ascomicetos/enzimologia , Ascomicetos/genética , Proteínas Fúngicas/genética , Oxirredutases/biossíntese , Oxirredutases/genéticaRESUMO
At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.
Assuntos
Biocatálise , Engenharia de Proteínas , Thermotoga maritima/enzimologia , alfa-Galactosidase/metabolismo , Glicosilação , Hidrólise , Modelos Moleculares , Mutação , Nitrofenilgalactosídeos/metabolismo , Thermotoga maritima/genética , alfa-Galactosidase/genéticaRESUMO
Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.
Assuntos
Aspergillus/enzimologia , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Dobramento de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Ácido Aspártico/química , Domínio Catalítico , Sequência Conservada , Frutose/metabolismo , Glutamina/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosilação , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Água/químicaRESUMO
Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.
Assuntos
Galactose/química , Penicillium/enzimologia , beta-Galactosidase/química , Sequência de Aminoácidos , Sítios de Ligação , Metabolismo dos Carboidratos , Cristalografia por Raios X , Galactose/metabolismo , Glicosilação , Dados de Sequência Molecular , Penicillium/metabolismo , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , beta-Galactosidase/metabolismoRESUMO
The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.
Assuntos
Galactose/metabolismo , Trichoderma/enzimologia , alfa-Galactosidase/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , alfa-Galactosidase/químicaRESUMO
A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.
Assuntos
Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/enzimologia , Gluconatos/metabolismo , Gluconobacter/enzimologia , L-Iditol 2-Desidrogenase/metabolismo , Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Gluconobacter/genética , Cinética , L-Iditol 2-Desidrogenase/genética , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/genética , Recombinação Genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
An exo-1,3-beta-glucanase has been isolated from cultural filtrate of T. viride AZ36. The N-terminal sequence of the purified enzyme (m = 61 +/- 1 kDa) showed no significant homology to other known glucanases. The 1,3-beta-glucanase displayed high activity against laminarins, curdlan, and 1,3-beta-oligoglucosides, but acted slowly on 1,3-1,4-beta-oligoglucosides. No significant activity was detected against high molecular mass 1,3-1,4-beta-glucans. The enzyme carried out hydrolysis with inversion of the anomeric configuration. Whereas only glucose was released from the nonreducing terminus during hydrolysis of 1,3-beta-oligoglucosides, transient accumulation of gentiobiose was observed during hydrolysis of laminarins. The gentiobiose was subsequently degraded to glucose. The Michaelis constants Km and Vmax have been determined for the hydrolysis of 1,3-beta-oligoglucosides with degrees of polymerization ranging from 2 to 6. Based on these data, binding affinities for subsites were calculated. Substrate binding site contained at least five binding sites for sugar residues.
Assuntos
Glucana 1,3-beta-Glucosidase , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Trichoderma/enzimologia , Sequência de Aminoácidos , Configuração de Carboidratos , Sequência de Carboidratos , Domínio Catalítico , Glucanos/química , Glicosídeo Hidrolases/genética , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligossacarídeos/química , Polissacarídeos/química , Estereoisomerismo , Especificidade por Substrato , Trichoderma/genéticaRESUMO
BACKGROUND: New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. METHODS: The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. RESULTS: All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed alpha-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside in the range of 10(-4) M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-alpha-D-glucopyranose. CONCLUSIONS: IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.
Assuntos
Amiloide/metabolismo , Imunoglobulina A Secretora/farmacologia , Imunoglobulina G/farmacologia , Leite Humano/enzimologia , Leite Humano/imunologia , Adulto , Amilases/análise , Amilases/metabolismo , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Feminino , Glucosidases/metabolismo , Humanos , Hidrólise , Immunoblotting , Imunoglobulina A Secretora/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Indicadores e Reagentes , Cinética , Maltose/química , Oligossacarídeos/metabolismo , Especificidade por SubstratoRESUMO
An alpha-L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving alpha1 --> 6-, alpha1 -->3-, alpha1 --> 4-, and alpha1 --> 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The alpha-L-fucosidase hydrolyzes p-nitrophenyl alpha-L-fucopyranoside with V(max) of 12.0 +/- 0.1 microM/min/mg and K(m) = 0.20 +/- 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 +/- 10 kDa consisting of four identical subunits with a molecular mass of 61.0 +/- 0.5 kDa. The N-terminal sequence showed homology to some alpha-L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl alpha-L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the alpha-L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl alpha-L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.
Assuntos
Thermus/enzimologia , alfa-L-Fucosidase/metabolismo , Sequência de Aminoácidos , Cromatografia/métodos , Sequência Conservada , Eletroforese em Gel de Poliacrilamida , Fucose/biossíntese , Fucose/química , Glicosilação , Cinética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Ultrafiltração , alfa-L-Fucosidase/isolamento & purificaçãoRESUMO
Crystals of an extracellular beta-galactosidase from Penicillium sp. (MW = 120 +/- 5 kDa) have been obtained from a sodium phosphate buffer using PEG as precipitant. The crystals belong to the tetragonal space group P4(1)or P4(3), with unit-cell parameters a = b = 110.82, c = 161.28 A, and diffract to 1.85 A resolution at a synchrotron source.
Assuntos
Penicillium/enzimologia , beta-Galactosidase/isolamento & purificação , Cristalização , Cristalografia por Raios X , Glicosilação , Conformação Proteica , beta-Galactosidase/química , beta-Galactosidase/metabolismoRESUMO
An extracellular multifunctional beta-xylosidase was purified from a culture of the fungus Trichoderma reesei. The active 95 +/- 5 kDa enzyme has been crystallized from sodium acetate buffer using PEG as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.75, b = 98.54, c = 227.25 A, and diffract beyond 2.7 A resolution. X-ray data were collected from frozen crystals on a synchrotron source.
Assuntos
Trichoderma/enzimologia , Xilosidases/química , Cristalização , Cristalografia por Raios X , Peso Molecular , Xilosidases/isolamento & purificaçãoRESUMO
The 160 kDa alpha-mannosidase (E.C. 3.2.1.24) isolated from culture filtrate of Trichoderma reesei has wide aglycon specificity but cleaves the alpha1 --> 2 and alpha1 --> 3 mannosidic bonds with higher rate than alpha1 --> 6 bond and slowly hydrolyses yeast mannan and 1,6-alpha-mannan. The specific activity of the enzyme and rate constant in the reaction with p-nitrophenyl-alpha-D-mannopyranoside were 0.15 U/mg and 1.62 x 10(-4) microM/min/microg, respectively, at optimal pH 6.5. We have found that in vitro enzyme is able to cleave off 30% of total alpha-mannopyranosyl residues from N- and O-linked glycans of secreted glycoproteins. The activity of the alpha-mannosidase toward glycoproteins in vivo was studied comparing the structures of O- and N-linked glycans of glycoproteins isolated from the cultures growing with and without 1-deoxymannojirimycin, an inhibitor of alpha-mannosidases. Difference in structures of these glycans may be explained by postsecretory deglycosylation catalysed by the alpha-mannosidase.
Assuntos
Glicoproteínas/metabolismo , Manosidases/metabolismo , Trichoderma/enzimologia , 1-Desoxinojirimicina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/metabolismo , Glicosilação , Cinética , Mananas/metabolismo , Manose/metabolismo , Especificidade por Substrato , alfa-ManosidaseRESUMO
Alpha-mannosidase was isolated from the culture liquid of Oerskovia sp. The purified enzyme had a molecular mass of 480 kDa and comprises four identical subunits. The enzyme cleaves bonds in side chains of yeast mannan (Km = 0.08 mM, k(cat) = 1.02 micromol x min(-1) x mg(-1)) and reveals a low activity towards p-nitrophenyl alpha-D-mannopyranoside. The alpha-mannosidase is a Ca2+-dependent enzyme and is inhibited by EDTA. The enzyme possess no endo-mannosidase activity releasing only mannose in the reaction with the inversion of anomeric configuration and could be classified as exo-alpha-mannanase. The enzyme revealed a high deglycosylating activity towards the short mannose-rich O-linked carbohydrate chains of glycoproteins.
Assuntos
Actinomycetales/enzimologia , Glicoproteínas/metabolismo , Manosidases/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Carboidratos/química , Glicoproteínas/química , Cinética , Mananas/química , Mananas/metabolismo , Manose/química , Manosidases/química , Manosidases/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Especificidade por Substrato , alfa-ManosidaseRESUMO
Alpha-galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by beta-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: alpha1-6 D-mannopyranosyl-alpha-D-glycopyranoside and alpha1-6 D-glucopyranosyl-alpha-D-galactopyranoside and one trimer: alpha-D-glucopyranosyl-alpha1-2 D-mannopyranosyl-alpha1-6 D-galactopyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo-beta-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo-beta-N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of alpha-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of alpha-galactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.
Assuntos
Trichoderma/enzimologia , alfa-Galactosidase/química , Sequência de Aminoácidos , Configuração de Carboidratos , Sequência de Carboidratos , Carboidratos/química , Desoxiglucose/farmacologia , Estabilidade Enzimática , Proteínas Fúngicas/química , Glicoproteínas/química , Glicosilação , Cinética , Dados de Sequência Molecular , Polissacarídeos/química , Trichoderma/genética , Tunicamicina/farmacologia , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoRESUMO
The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.