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Edward A. Bayer - One of the best experts on this subject based on the ideXlab platform.
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Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode
2018Co-Authors: Kun-di Zhang, Edward A. Bayer, Ye-fei Wang, Yan-lin Zheng, Fang-cheng Tan, Li-shan Yao, Lu-shan WangAbstract:Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei Exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal Exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite −4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases
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modular organization of the thermobifida fusca Exoglucanase cel6b impacts cellulose hydrolysis and designer cellulosome efficiency
Biotechnology Journal, 2017Co-Authors: Eva Setterlamed, Johanna Stern, Sarah Moraïs, Raphael Lamed, Edward A. BayerAbstract:: Cellulose deconstruction can be achieved by three distinct enzymatic paradigms: free enzymes, multifunctional enzymes, and self-assembled, multi-enzyme complexes (cellulosomes). To study their comparative efficiency, the simple and efficient cellulolytic system of the aerobic bacterium, Thermobifida fusca, is developed as an enzymatic model. In previous studies, most of its cellulases are successfully converted to the cellulosomal mode and exhibited high cellulolytic activities, except for Cel6B, a key Exoglucanase of the T. fusca enzymatic system. Here, the impact of the modular organization of Cel6B on enzymatic activity is investigated. The position of the cellulose-binding module (CBM), its family and linker segment are shown to affect activity. Surprisingly, exchange of the native family-2 CBM to family-3 generates an increase in Cel6B activity on cellulosic substrates. Conversion of Cel6B to the cellulosomal mode by fusing a cohesin to the catalytic module enables formation of divalent enzyme complexes with dockerin-bearing enzymes. The resultant pseudo-cellulosomes, containing Cel6B combined with endoglucanase Cel5A, exhibits enhanced enzymatic activity, compared to mixtures of wild-type enzymes or bifunctional enzymes, unlike similar pseudo-cellulosomes containing endoglucanase Cel6A or proccessive endoglucanase Cel9A. Insight into the different enzymatic paradigms benefits ongoing development of efficient cellulolytic systems for conversion of plant-derived biomass into valuable sugars. NOVELTY STATEMENT: The protein engineering of the modular arrangement of a key Exoglucanase from a highly cellulolytic bacterium, Thermobifida fusca, served to explore and compare three major enzymatic paradigms for cellulose degradation. This approach revealed highly active chimaeric forms of the Exoglucanase that act in synergy together with a potent endoglucanase in bifunctional enzymes or divalent pseudo-cellulosome-like complexes. Such engineered enzymes could be further integrated into larger enzymatic complexes, thereby providing a significant step forward towards conversion of the entire T. fusca free cellulolytic system into the cellulosomal modex and the enhanced conversion of cellulosic biomass into soluble sugars.
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Thermobifida fusca Exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A.
Systems and Synthetic Biology, 2010Co-Authors: Jonathan Caspi, Raphael Lamed, Diana C. Irwin, David Wilson, Yoav Barak, Rachel Haimovitz, Hadar Gilary, Edward A. BayerAbstract:Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and Exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six Exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.
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Enhanced cellulose degradation by nano-complexed enzymes: Synergism between a scaffold-linked Exoglucanase and a free endoglucanase.
Journal of Biotechnology, 2010Co-Authors: Sarah Moraïs, Raphael Lamed, Jonathan Caspi, Arnon Heyman, Yoav Barak, David B Wilson, Oded Shoseyov, Edward A. BayerAbstract:Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populustremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosomal cohesin) modules. In the present work, we have displayed a potent Exoglucanase (Cel6B) from the aerobic cellulolytic bacterium, Thermobifida fusca, on a cohesin-bearing SP1 scaffold. For this purpose, a chimaeric form of the enzyme, fused to a cellulosomal dockerin module, was prepared. Full incorporation of 12 dockerin-bearing Exoglucanase molecules onto the cohesin-bearing scaffold was achieved. Cellulase activity was tested on two cellulosic substrates with different levels of crystallinity, and the activity of the scaffold-linked Exoglucanase was significantly reduced, compared to the free dockerin-containing enzyme. However, addition of relatively low concentrations of a free wild-type endoglucanase (T. fusca Cel5A) that bears a cellulose-binding module, in combination with the complexed Exoglucanase resulted in a marked rise in activity on both cellulosic substrates. The endoglucanase cleaves internal sites of the cellulose chains, and the new chain ends of the substrate were now readily accessible to the scaffold-borne Exoglucanase, thereby resulting in highly effective, synergistic degradation of cellulosic substrates.
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Conversion of Thermobifida fusca free Exoglucanases into cellulosomal components: Comparative impact on cellulose-degrading activity
Journal of Biotechnology, 2008Co-Authors: Jonathan Caspi, Raphael Lamed, Diana C. Irwin, Henri-pierre Fierobe, David Wilson, Edward A. BayerAbstract:Cellulosomes are multi-enzyme complexes produced by certain anaerobic bacteria that exhibit efficient degradation of plant cell wall polysaccharides. To understand their enhanced levels of hydrolysis, we are investigating the effects of converting a free-cellulase system into a cellulosomal one. To achieve this end, we are replacing the cellulose-binding module of the native cellulases, produced by the aerobic bacterium Thermobifida fusca, with a cellulosome-derived dockerin module of established specificity, to allow their incorporation into defined “designer cellulosomes”. In this communication, we have attached divergent dockerins to the two Exoglucanases produced by T. fusca Exoglucanase, Cel6B and Cel48A. The resultant fusion proteins were shown to bind efficiently and specifically to their matching cohesins, and their activities on several different cellulose substrates were compared. The lack of a cellulose-binding module in Cel6B had a deleterious effect on its activity on crystalline substrates. In contrast, the dockerin-bearing family-48 Exoglucanase showed increased levels of hydrolytic activity on carboxymethyl cellulose and on both crystalline substrates tested, compared to the wild-type enzyme. The marked difference in the response of the two Exoglucanases to incorporation into a cellulosome, suggests that the family-48 cellulase is more appropriate than the family-6 enzyme as a designer cellulosome component.
Germán Larriba - One of the best experts on this subject based on the ideXlab platform.
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The major Exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation.
Biomolecular Engineering, 2001Co-Authors: Germán Larriba, Rosario CuevaAbstract:The major yeast Exoglucanase (ExgIb) consists of a 408 amino acid polypeptide carrying two short N-linked oligosaccharides attached to asparagines 165 (Asn(165)) and 325 (Asn(325)). These oligosaccharides are very similar, in both length and composition, to those present in the vacuolar protease carboxypeptidase Y. Minor glycoforms of Exoglucanase arise by underglycosylation of the protein precursor (Exg(165) and Exg(325)) or by elongation of the second oligosaccharide (ExgIa). The fact that these glycoforms can be readily separated and identified by HPLC and/or Western blots converts ExgI in an excellent model to study the role of the several components or branches of the precursor oligosaccharide in the efficiency and selectivity of the oligosaccharidyl transferase in vivo. We have found that the presence of a single glucose attached to Dol-PP-GlcNAc(2)-Man(9) increases the efficiency of transfer of that oligosaccharide to the protein acceptor. Also, the glucotriose unit appears to be involved in the selection of the sequons to be occupied, in such a way that its absence results in a bias towards the glycosylation of a particular sequon. Finally, we have shown the transfer of GlcNAc(2) from Dol-PP-GlcNAc(2) to Exoglucanase, an indication that this intermediate is able to translocate from the cytoplasmic to the lumenal face of the endoplasmic reticulum membrane.
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N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast Exoglucanase.
Yeast, 1998Co-Authors: Rosario Cueva, Cecilio Cotano, Germán LarribaAbstract:Transfer of truncated oligosaccharides to yeast Exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the Exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had aYnity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N 4 -N-acetyl-‚-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PPGlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum. ? 1998 John Wiley & Sons, Ltd.
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In vivo processing of the precursor of the major Exoglucanase by KEX2 endoprotease in the Saccharomyces cerevisiae secretory pathway.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1996Co-Authors: Ricardo D. Basco, Encarnación Andaluz, Rosario Cueva, Germán LarribaAbstract:Abstract We have established the main post-translational modification of the major Exoglucanase of Saccharomyces cerevisiae as the enzyme progresses through the secretory pathway. The protein portion of the enzyme accumulated by sec18 cells was about 2 kDa larger than that of the secreted enzyme. This precursor (form A) was stable when maintained in the endoplasmic reticulum but was processed to the mature form (form B) before the block imposed by the sec7 mutation. Sec7 cells, when incubated at 37°C, accumulated form B first, but upon prolonged incubation, form A was preferentially accumulated. When the supply of newly synthesized Exoglucanase was prevented by the addition of cycloheximide, the accumulated A was transformed into B in the presence of altered Sec7p that still prevented secretion. Conversion of A into B was prevented in the double mutant sec7 kex2-1 , indicating that Kex2p is central to the in vivo processing. Consistent with this, a KEX2 deletion mutant secreted form A exclusively. Conversion of A into B was also prevented in sec7 cells by the presence of dinitrophenol, a poison that depletes ATP levels, indicating that processing is dependent upon intracellular transport which involves ER → Golgi and/or, at least, one intra-Golgi step(s). It follows that this transport step(s) is independent of functional Sec7p.
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Reduced efficiency in the glycosylation of the first sequon of Saccharomyces cerevisiae Exoglucanase leads to the synthesis and secretion of a new glycoform of the molecule
Yeast, 1993Co-Authors: Ricardo D. Basco, Luis M. Hernández, M. Dolores Muñoz, Carlos R. Vázquez De Aldana, Germán LarribaAbstract:In addition to Exoglucanases (EXGs) I and II, old cultures of Saccharomyces cerevisiae secreted into the culture medium a new immunologically-related material that exhibited Exoglucanase activity. The new Exoglucanase (EXGII1/2) was purified from stationary-phase cultures. It turned out to be a glycoprotein whose protein portion was identical to that of the other two isoenzymes in terms of ionic properties, size, amino acid composition and NH2-terminal sequence (25 residues). Disruption of the structural gene encoding EXGs I and II resulted in a strain unable to secrete all three isoenzymes. EXGII1/2 was indistinguishable in terms of molecular weight from the single intermediate detected during the deglycosylation (mediated by endo H) of EXGII by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, the new isoenzyme contains only one of the two slightly elongated mannan inner cores present in enzyme II. Two intermediates were, however, detected when the deglycosylation of EXGII was monitored by ion-exchange chromatography (high-pressure liquid chromatography). Site-directed mutagenesis indicated that the major intermediate, which eluted at about the same position as enzyme II1/2, corresponded to protein molecules carrying the oligosaccharide attached to the Asn of the second sequon, whereas the minor one carried the oligosaccharide in the first potential glycosylation site. Several lines of evidence indicate that EXGII1/2 is a biosynthetic product resulting from an imbalance between the rate of protein synthesis and the glycosylation capabilities of the glycosylation machinery.
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The major Exoglucanase from Candida albicans: A non-glycosylated secretory monomer related to its counterpart from Saccharomyces cerevisiae
Yeast, 1991Co-Authors: Juan P. Luna‐arias, Encarnación Andaluz, Juan C. Ridruejo, Isabel Olivero, Germán LarribaAbstract:Exoglucanases secreted by two different strains from Candida albicans have been purified to homogeneity. The purified enzyme from each strain behaved as a non-glycosylated monomer (molecular weight 38,000) that was identical in terms of sodium dodecyl sulphate/polyacrylamide gel electrophoresis comigration, amino acid analysis and amino terminal sequence. The amino acid composition was similar to that of the major Exoglucanase from Saccharomyces cerevisiae. In addition, these two enzymes displayed a 50% homology in the first 35 amino acids of the amino terminus. Antibodies against the deglycosylated Exoglucanase (treated with Endo H) from S. cerevisiae were reactive with the Exoglucanase from C. albicans and vice versa. Immunoblotting proved to be a semiquantitative method to detect C. albicans antigen in culture fluids. The Exoglucanase from C. albicans appears to enter the secretory pathway without undergoing N-glycosylation.
Madihah M S - One of the best experts on this subject based on the ideXlab platform.
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production of cellulases and xylanase by aspergillus fumigatus sk1 using untreated oil palm trunk through solid state fermentation
Process Biochemistry, 2013Co-Authors: S K Ang, E M Shaza, Y Adibah, Suraini A A, Madihah M SAbstract:Abstract Direct utilization of untreated oil palm trunk (OPT) for cellulases and xylanase production by Aspergillus fumigatus SK1 was conducted under solid-state fermentation (SSF). The highest activities of extracellular cellulases and xylanases were produced at 80% moisture level, initial pH 5.0, 1 × 10 8 spore/g (inoculum) with 125 μm of OPT as sole carbon source. The cellulases and xylanase activities obtained were 54.27, 3.36, 4.54 and 418.70 U/g substrates for endoglucanase (CMCase), Exoglucanase (FPase), β-glucosidase and xylanase respectively. The crude cellulases and xylanase required acidic condition to retain their optimum activities (pH 4.0). Crude cellulases and xylanase were more stable at 40 °C compared to their optimum activities conditions (60 °C for FPase and 70 °C for CMCase, β-glucosidase and xylanase). SDS-PAGE and zymogram analysis showed that Aspergillus fumigatus SK1 could secrete cellulases (endoglucanase, Exoglucanase and β-glucosidase), xylanase and protease. Enzymatic degradation of alkaline treated OPT with concentrated crude cellulases and xylanases resulted in producing polyoses.
Rajan Sankaranarayanan - One of the best experts on this subject based on the ideXlab platform.
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A mutation in an Exoglucanase of Xanthomonas oryzae pv. oryzae, which confers an endo mode of activity, affects bacterial virulence, but not the induction of immune responses, in rice.
Molecular Plant Pathology, 2017Co-Authors: Lavanya Tayi, Sushil Kumar, Rajkanwar Nathawat, Asfarul S. Haque, Roshan V. Maku, Hitendra Kumar Patel, Rajan Sankaranarayanan, Ramesh V. SontiAbstract:Summary Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall degrading enzymes which includes cellulases, xylanases, pectinases to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo but is also a potent inducer of innate immune responses of rice. In this study, we have solved the crystal structure of the catalytic domain of CbsA protein to a resolution of 1.86A. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N and C-terminal loops enclosing the active site which is a characteristic structural feature of an Exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in catalysis and biological functions of CbsA. Intriguingly, a D131A CbsA mutant protein displayed enzymatic activity of a typical endoglucanase. The D131A CbsA is as proficient as wild-type (Wt) CbsA in inducing rice immune responses but is deficient in virulence promoting activity. This indicates that the specific Exoglucanase activity of Wt CbsA protein is required for this protein to promote growth of Xoo in rice. This article is protected by copyright. All rights reserved.
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Crystallization and preliminary crystallographic studies of CbsA, a secretory Exoglucanase from Xanthomonas oryzae pv. oryzae.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2012Co-Authors: Sushil Kumar, Asfarul S. Haque, Ramesh V. Sonti, Gopaljee Jha, Rajan SankaranarayananAbstract:The bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. The secreted Exoglucanase CbsA is an important virulence factor of this pathogen. It belongs to the glycosyl hydrolase 6 family of proteins based on the carbohydrate-active enzyme (CAZY) classification. In this study, CbsA has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.86 A and belonged to space group P212121. It contained one monomer per asymmetric unit, with a solvent content of 45.8%.
Ricardo D. Basco - One of the best experts on this subject based on the ideXlab platform.
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In vivo processing of the precursor of the major Exoglucanase by KEX2 endoprotease in the Saccharomyces cerevisiae secretory pathway.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1996Co-Authors: Ricardo D. Basco, Encarnación Andaluz, Rosario Cueva, Germán LarribaAbstract:Abstract We have established the main post-translational modification of the major Exoglucanase of Saccharomyces cerevisiae as the enzyme progresses through the secretory pathway. The protein portion of the enzyme accumulated by sec18 cells was about 2 kDa larger than that of the secreted enzyme. This precursor (form A) was stable when maintained in the endoplasmic reticulum but was processed to the mature form (form B) before the block imposed by the sec7 mutation. Sec7 cells, when incubated at 37°C, accumulated form B first, but upon prolonged incubation, form A was preferentially accumulated. When the supply of newly synthesized Exoglucanase was prevented by the addition of cycloheximide, the accumulated A was transformed into B in the presence of altered Sec7p that still prevented secretion. Conversion of A into B was prevented in the double mutant sec7 kex2-1 , indicating that Kex2p is central to the in vivo processing. Consistent with this, a KEX2 deletion mutant secreted form A exclusively. Conversion of A into B was also prevented in sec7 cells by the presence of dinitrophenol, a poison that depletes ATP levels, indicating that processing is dependent upon intracellular transport which involves ER → Golgi and/or, at least, one intra-Golgi step(s). It follows that this transport step(s) is independent of functional Sec7p.
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Molecular biology of yeast Exoglucanases
FEMS Microbiology Letters, 1995Co-Authors: Germán Larriba, Rosario Cueva, Encarnación Andaluz, Ricardo D. BascoAbstract:Three Exoglucanase (Exg) genes have been reported in Saccharomyces cerevisiae. Gene EXG1 encodes the major isoenzyme (Exgl). Differential glycosylation of the primary translation product throughout the secretory pathway results in the secretion of several glycoforms. The major glycoform (Exglb) contains two short carboxypeptidase Y-like oligosaccharides attached to both potential glycosylation sites present in the molecule. A minor glycoform (Exgla) arises from the former by elongation of the second oligosaccharide. The protein portion is processed in the secretory pathway by the Kex2 protease. Gene EXG2 encodes a 63 kDa polypeptide with 12 potential glycosylation sites. The predicted protein, Exgll, carries a signal peptide at the amino terminus and a glycosyl-phosphatidyl inositol anchoring motif at the carboxyl end. The latter appears responsible for the particulate nature of this isoenzyme, since its elimination results in the secretion of this activity into the culture medium. Gene SSG1 encodes a 52 kDa polypeptide which is specifically synthesized during sporulation of diploids. SSC1 expression is under control of both sexual (a1-α2 element) and nutritional control. Although homozygous ssg1 / ssg1 diploid strains are still able to complete sporulation, they exhibited a delay in the appearance of mature asci. Single or double disruption of EXG1 and EXG2 did not result in any relevant phenotype and the triple mutant behaved as ssg1 /ssg1. A Exgl-related enzyme is secreted by Candida albicans. All these four enzymes share 8 highly conserved regions in the same relative positions, indicating that they derive from a common ancestor. However, no clear function has so far been demonstrated for them.
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Yeast Exoglucanases. Where redundancy implies necessity.
Archives of medical research, 1993Co-Authors: G Larriba, Ricardo D. Basco, E. Andaluz, J. P. Luna-ariasAbstract:Three Exoglucanase genes have been described in Saccharomyces cerevisiae. The bulk of the Exoglucanase (EXO) activity is encoded by the EXG1 gene, whose primary gene product is differentially glycosylated during its transit to the cell surface to yield three isoenzymes: EXOI, EXOII and EXOII1/2. EXOII, the major isoenzyme, carries two short oligosaccharides, each one consisting of an inner core with a single branch of the outer chain, attached to both potential glycosylation sites present in the molecule (Asn165 and Asn325). EXOI and EXOII1/2 are minor representatives. The second carries a single short sugar residue attached to Asn165 whereas the former elongate the outer chain of, at least, one oligosaccharide as other cell wall mannoproteins. The protein portion of the EXGI gene product is cleaved in Golgi by the Kex2 protease. A different Exoglucanase, encoded by a second gene (EXG2), has been characterized as a heavily glycosylated, membrane bound 200 kDa glycoprotein. Finally, a third Exoglucanase, encoded by the SSG gene, is synthesized during sporulation of diploids. Exoglucanases similar to those encoded by the EXG1 gene have been detected in other yeasts and characterized in depth in Candida albicans. The three polypeptides from S. cerevisiae and its counterpart from C. albicans have several conserved regions occupying the same relative positions. Studies on the function of these highly conserved enzymes are rather inconclusive.
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Reduced efficiency in the glycosylation of the first sequon of Saccharomyces cerevisiae Exoglucanase leads to the synthesis and secretion of a new glycoform of the molecule
Yeast, 1993Co-Authors: Ricardo D. Basco, Luis M. Hernández, M. Dolores Muñoz, Carlos R. Vázquez De Aldana, Germán LarribaAbstract:In addition to Exoglucanases (EXGs) I and II, old cultures of Saccharomyces cerevisiae secreted into the culture medium a new immunologically-related material that exhibited Exoglucanase activity. The new Exoglucanase (EXGII1/2) was purified from stationary-phase cultures. It turned out to be a glycoprotein whose protein portion was identical to that of the other two isoenzymes in terms of ionic properties, size, amino acid composition and NH2-terminal sequence (25 residues). Disruption of the structural gene encoding EXGs I and II resulted in a strain unable to secrete all three isoenzymes. EXGII1/2 was indistinguishable in terms of molecular weight from the single intermediate detected during the deglycosylation (mediated by endo H) of EXGII by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, the new isoenzyme contains only one of the two slightly elongated mannan inner cores present in enzyme II. Two intermediates were, however, detected when the deglycosylation of EXGII was monitored by ion-exchange chromatography (high-pressure liquid chromatography). Site-directed mutagenesis indicated that the major intermediate, which eluted at about the same position as enzyme II1/2, corresponded to protein molecules carrying the oligosaccharide attached to the Asn of the second sequon, whereas the minor one carried the oligosaccharide in the first potential glycosylation site. Several lines of evidence indicate that EXGII1/2 is a biosynthetic product resulting from an imbalance between the rate of protein synthesis and the glycosylation capabilities of the glycosylation machinery.
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Two glycosylation patterns for a single protein (Exoglucanase) in Saccharomyces cerevisiae
FEMS Microbiology Letters, 1990Co-Authors: Manuel Ra'irez, Luis M. Hernández, M. Dolores Muñoz, Ricardo D. Basco, Guillermo Giménez-gallego, Germán LarribaAbstract:Exoglucanases (β-glucosidases) I and II secreted into the culture medium by Saccharomyces cerevisiae were purified from cell cultures harvested at the early exponential phase of growth in order to avoid contamination of the second by a new immunologically-related material. The amino acid composition of the purified enzymes was roughly the same. In addition, both Exoglucanases exhibited an identical NH2-terminal sequence (50 residues). These results confirm our previous results about the identity of the protein moieties of both enzymes. Exoglucanase I appears to arise by elongation of one or both short oligosaccharides present in enzyme II.