RESUMEN
Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted ß-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Análisis de Secuencia de ADN , Aspergillus nidulans/enzimología , Bifidobacterium adolescentis/enzimología , Carbohidratos/análisis , Electroforesis , Colorantes Fluorescentes , Límite de Detección , Metagenómica , Podospora/enzimología , Especificidad por SustratoRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Polisacáridos/metabolismo , Cromatografía por Intercambio Iónico , Vectores Genéticos , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/aislamiento & purificación , Mutagénesis Sitio-Dirigida , Pichia/genéticaRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment.
Asunto(s)
Proteínas Bacterianas/química , Disulfuros/química , Oxigenasas de Función Mixta/química , Modelos Moleculares , Programas Informáticos , Streptomyces coelicolor/enzimología , Estabilidad de Enzimas , CalorRESUMEN
The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. ß-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the ß-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the ß-glucosidase from H. jecorina (HjCel3A) with the ß-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 ß-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.
Asunto(s)
Eurotiales/enzimología , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Cristalografía por Rayos X , Eurotiales/química , Eurotiales/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glicosilación , Hidrólisis , Hypocrea/química , Hypocrea/enzimología , Hypocrea/metabolismo , Lignina/metabolismo , Modelos Moleculares , Conformación Proteica , Multimerización de ProteínaRESUMEN
The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Oxigenasas de Función Mixta/biosíntesis , Pichia/metabolismo , Trichoderma/enzimología , Secuencia de Aminoácidos , Biomasa , Celulosa/química , Celulosa/genética , ADN de Hongos/genética , Fermentación , Proteínas Fúngicas/genética , Hidrólisis , Factor de Apareamiento , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Polisacáridos/química , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Trichoderma/genéticaRESUMEN
UNLABELLED: We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca(2+) takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca(2+). These results unify the generally accepted lectin hypothesis with the historically first-proposed "Ca(2+)-bridge" hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating. IMPORTANCE: Yeast cells can form multicellular clumps under adverse growth conditions that protect cells from harsh environmental stresses. The floc formation is based on the self-interaction of Flo proteins via an N-terminal PA14 lectin domain. We have focused on the flocculation mechanism and its role. We found that carbohydrate specificity and affinity are determined by the accessibility of the binding site of the Flo proteins where the external loops in the ligand-binding domains are involved in glycan recognition specificity. We demonstrated that, in addition to the Flo lectin-glycan interaction, glycan-glycan interactions also contribute significantly to cell-cell recognition and interaction. Additionally, we show that flocculation provides a uniquely organized multicellular ultrastructure that is suitable to induce and accomplish cell mating. Therefore, flocculation is an important mechanism to enhance long-term yeast survival.
Asunto(s)
Adhesión Celular , Conjugación Genética , Floculación , Lectinas de Unión a Manosa/metabolismo , Viabilidad Microbiana , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Perfilación de la Expresión Génica , Lectinas de Unión a Manosa/química , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos/análisis , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Análisis de Secuencia de ADNRESUMEN
In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.
Asunto(s)
Proteínas Fúngicas/química , Hypocrea/enzimología , Liasas/química , Secuencia de Aminoácidos , Calcio/química , Dominio Catalítico , Complejos de Coordinación/química , Cristalografía por Rayos X , Glicol de Etileno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Desplegamiento ProteicoRESUMEN
Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Celulosa/metabolismo , Proteínas Fúngicas/metabolismo , Lignina/metabolismo , Sitios de Unión , Unión Competitiva , Biocatálisis , Dominio Catalítico , Celulosa 1,4-beta-Celobiosidasa/química , Proteínas Fúngicas/química , Glicosilación , Hidrólisis , Espectrometría de Masas , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Trichoderma/enzimología , Trichoderma/metabolismoRESUMEN
Endo-N-acetyl-ß-D-glucosaminidases (ENGases) hydrolyze the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. The endo-N-acetyl-ß-D-glucosaminidases classified into glycoside hydrolase family 18 are small, bacterial proteins with different substrate specificities. Recently two eukaryotic family 18 deglycosylating enzymes have been identified. Here, the expression, purification and the 1.3Å resolution structure of the ENGase (Endo T) from the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei) are reported. Although the mature protein is C-terminally processed with removal of a 46 amino acid peptide, the protein has a complete (ß/α)8 TIM-barrel topology. In the active site, the proton donor (E131) and the residue stabilizing the transition state (D129) in the substrate assisted catalysis mechanism are found in almost identical positions as in the bacterial GH18 ENGases: Endo H, Endo F1, Endo F3, and Endo BT. However, the loops defining the substrate-binding cleft vary greatly from the previously known ENGase structures, and the structures also differ in some of the α-helices forming the barrel. This could reflect the variation in substrate specificity between the five enzymes. This is the first three-dimensional structure of a eukaryotic endo-N-acetyl-ß-D-glucosaminidase from glycoside hydrolase family 18. A glycosylation analysis of the cellulases secreted by a Hypocrea jecorina Endo T knock-out strain shows the in vivo function of the protein. A homology search and phylogenetic analysis show that the two known enzymes and their homologues form a large but separate cluster in subgroup B of the fungal chitinases. Therefore the future use of a uniform nomenclature is proposed.
Asunto(s)
Celulasa/metabolismo , Proteínas Fúngicas/química , Hypocrea/enzimología , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inactivación de Genes , Glicosilación , Hypocrea/genética , Hypocrea/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteoma/metabolismo , Homología Estructural de Proteína , Zinc/químicaRESUMEN
Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of ß-sheets. The binding of N-Flo1p to D-mannose, α-methyl-D-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.
Asunto(s)
Mananos/metabolismo , Lectinas de Unión a Manosa/metabolismo , Manosa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Floculación , Glicosilación , Lectinas de Unión a Manosa/química , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de Fluorescencia , VolumetríaRESUMEN
An enzyme with mannosyl glycoprotein endo-N-acetyl-beta-D-glucosaminidase (ENGase)-type activity was partially purified from the extracellular medium of the mould Hypocrea jecorina (Trichoderma reesei). Internal peptides were generated and used to identify the gene in the T. reesei genome. The active enzyme is processed both at the N- and at the C-terminus. High-mannose-type glycoproteins are good substrates, whereas complex-type glycans are not hydrolysed. The enzyme represents the first fungal member of glycoside hydrolase family 18 with ENGase-type activity. Bacterial ENGases and the fungal chitinases belonging to the same family show very low homology with Endo T. Database searches identify several highly homologous genes in fungi and the activity is also found within other Trichoderma species. This ENGase activity, not coregulated with cellulase production, could be responsible for the extensive N-deglycosylation observed for several T. reesei cellulases.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocrea/enzimología , Hypocrea/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Análisis por Conglomerados , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Procesamiento Proteico-Postraduccional , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
N-Glycosylation of cellobiohydrolase I from the fungus Trichoderma reesei (strain Rut-C30) is studied using a combination of electrophoretic, chromatographic and mass spectrometric techniques. As four potential N-glycosylation sites and several uncharged and phosphorylated high-mannose glycans are present, a large number of glycoforms and phospho-isoforms can be expected. Isoelectric focusing both in gel and in capillary format was successfully applied for the separation of the phospho-isoforms. They were extracted in their intact form from the gel and subsequently analysed by nanospray-Q-TOF-MS, thereby making use of a powerful two-dimensional technique. Nano-LC/MS/MS on a Q-Trap MS further allowed the determination of the glycosylation sites. As a novel approach, an oxonium ion was used in precursor ion scanning for selective detection of glycopeptides containing phosphorylated high-mannose glycans.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/química , Cromatografía Liquida/métodos , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Trichoderma/enzimología , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Focalización Isoeléctrica , Datos de Secuencia Molecular , NanotecnologíaRESUMEN
A capillary electrophoresis-mass spectrometric (CE-MS) method is described for the simultaneous analysis of uncharged and charged glycans. The glycans were labeled with the negatively charged tag 8-aminopyrene-1,3,6-trisulfonate by reductive amination and separated in an ammonium acetate buffer. A Q-Trap instrument was used for mass spectrometric detection. The CE-MS method was first optimized using maltooligosaccharides and ribonuclease B N-glycans and then applied to the characterization of enzymatically released N-glycans from the glycoprotein cellobiohydrolase I. The method, as developed, allowed differentiation of phosphorylated isomers and MS/MS provided useful structural information. Further structural evidence was obtained by studying the methylated glycans in off-line ESI-MS/MS experiments and by using a combination of chemical and enzymatic sequencing.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/análisis , Celulosa 1,4-beta-Celobiosidasa/química , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Polisacáridos/análisis , Polisacáridos/química , Conformación de Carbohidratos , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Iones/química , Isoenzimas/análisis , Isoenzimas/química , Isoenzimas/metabolismo , Fosforilación , Polisacáridos/metabolismoRESUMEN
The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions. As shown by ESI-MS and PAG-IEF analyses of both intact protein and the isolated catalytic core module, the microheterogeneity originates mainly from the variable ratio of single N-acetylglucosamine over high-mannose structures on the three N-glycosylation sites and from the presence or absence of phosphate residues. Fully N- and O-glycosylated Cel7A can only be isolated from minimal medium and probably reflects the initial complexity of the protein on leaving the glycosynthetic pathway. Extracellular activities are responsible for postsecretorial modifications in other cultivation conditions: alpha-(1-->2)-mannosidase, alpha-(1-->3)-glucosidase and an Endo H type activity participate in N-deglycosylation (core), whereas a phosphatase and a mannosidase are probably responsible for hydrolysis of O-glycans (linker). The effects are most prominent in corn steep liquor-enriched media, where the pH is closer to the pH optimum (5-6) of these extracellular hydrolases. In minimal medium, the low pH and the presence of proteases could explain for the absence of such activities. On the other hand, phosphodiester linkages in the catalytic module are only observed under specific conditions. The extracellular trigger is still unknown, but mannophosphorylation may be regulated intracellularly by alpha-(1-->2)-mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Trichoderma/enzimología , Dominio Catalítico , Medios de Cultivo , Líquido Extracelular/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Polisacáridos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Trichoderma/crecimiento & desarrolloRESUMEN
A systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (Cel7A or CBH I) isolated from several Trichoderma reesei strains grown in minimal media was performed. Using a combination of chromatographic, electrophoretic, and mass spectrometric methods, the presence of glucosylated and phosphorylated oligosaccharides on the three N-glycosylation sites of Cel7A core protein (from T. reesei strains Rut-C30 and RL-P37) confirms previous findings. With N-glycans isolated from other strains, no end-capping glucose could be detected. Phosphodiester linkages were however found in proteins from each strain and these probably occur on both the alpha1-3 and the alpha1-6 branch of the high-mannose oligosaccharide tree. Evidence is also presented for the occurrence of mannobiosyl units on the phosphodiester linkage. Therefore the predominant N-glycans on Cel7A can be represented as (ManP)(0-1)GlcMan(7-8)GlcNAc2 for the hyperproducing Rut-C30 and RL-P37 mutants and as (Man(1-2)P)(0-1-2)Man(5-6-7)GlcNAc2 for the wild-type strain and the other mutants. As shown by ESI-MS, random substitution of these structures on the N-glycosylation sites explains the heterogeneous glycoform population of the isolated core domains. PAG-IEF separates up to five isoforms, resulting from posttranslational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical for Trichoderma sp. and that, in the case of the Rut-C30 and RL-P37 strains, the presence of an end-capped glucose residue at the alpha1-3 branch apparently hinders a second mannophoshoryl transfer.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Trichoderma/enzimología , Secuencia de Carbohidratos , Dominio Catalítico , Celulosa 1,4-beta-Celobiosidasa/aislamiento & purificación , Cromatografía Liquida , Electroforesis , Electroforesis en Gel de Poliacrilamida , Glicosilación , Focalización Isoeléctrica , Datos de Secuencia Molecular , Mutación , Fosforilación , Polisacáridos/análisis , Procesamiento Proteico-Postraduccional , Especificidad de la Especie , Espectrometría de Masa por Ionización de Electrospray , Trichoderma/genéticaRESUMEN
The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
Asunto(s)
Celulasa/química , Hypocrea/metabolismo , Trichoderma/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Asparagina/química , Sitios de Unión , Dominio Catalítico , Celulasas/química , Cromatografía Líquida de Alta Presión , Endopeptidasas/química , Glicoproteínas/química , Glicósido Hidrolasas/química , Glicosilación , Hidrólisis , Focalización Isoeléctrica , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Unión Proteica , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray , alfa-Manosidasa/químicaRESUMEN
Xylanases are generally classified into glycosyl hydrolase families 10 and 11 and are found to frequently have an inverse relationship between their pI and molecular mass values. However, we have isolated a psychrophilic xylanase that belongs to family 8 and which has both a high pI and high molecular mass. This novel xylanase, isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis, is not homologous to family 10 or 11 enzymes but has 20-30% identity with family 8 members. NMR analysis shows that this enzyme hydrolyzes with inversion of anomeric configuration, in contrast to other known xylanases which are retaining. No cellulase, chitosanase or lichenase activity was detected. It appears to be functionally similar to family 11 xylanases. It hydrolyzes xylan to principally xylotriose and xylotetraose and is most active on long chain xylo-oligosaccharides. Kinetic studies indicate that it has a large substrate binding cleft, containing at least six xylose-binding subsites. Typical psychrophilic characteristics of a high catalytic activity at low temperatures and low thermal stability are observed. An evolutionary tree of family 8 enzymes revealed the presence of six distinct clusters. Indeed classification in family 8 would suggest an (alpha/alpha)(6) fold, distinct from that of other currently known xylanases.