Vladimír Farkaš - Academia.edu (original) (raw)

Papers by Vladimír Farkaš

Biochemical Journal, 1998

Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominan... more Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominantly by transglycosylation. In this process, fragments of cleaved polysaccharide are preferentially transferred to other xyloglucan molecules or their oligosaccharide subunits, with overall retention of the anomeric configuration of the glycosidic bond. In accordance with the theory, we propose that the cleavage and re-formation of the glycosidic bond in xyloglucan involves the formation of a glycosyl-enzyme intermediate which decomposes by transfer of the glycosyl moiety to a suitable carbohydrate acceptor. XETs from nasturtium seed cotyledons, mung bean hypocotyls and cauliflower florets interacted with xyloglucan to form complexes of high Mr as judged by gel-permeation chromatography. The nasturtium enzyme also showed evidence of XET-xyloglucan complex-formation according to anion-exchange chromatography and adsorption of the complex to filter paper on the basis of affinity of its xylo...

General physiology and biophysics, 2000

Plant xyloglucan endotransglycosylase (XET, EC 2.4.1.207) degrades its substrate by a transglycos... more Plant xyloglucan endotransglycosylase (XET, EC 2.4.1.207) degrades its substrate by a transglycosylation mechanism while endo-cleaving xyloglucan (XG) molecules at their beta-1,4-linked polyglucosyl main chain and transferring the newly generated reducing chain ends to hydroxyls at C-4 of non-reducing glucosyl ends of the main chains of other XG molecules or of low-Mr XG-fragments (OS). Kinetic data obtained with purified nasturtium seed (Tropaeolum majus, L.) XET while using high-Mr xyloglucan and 3H-labeled XGOS alditols (DP 7-9) as substrates could be best fitted to the model for Ping-Pong Bi Bi reaction mechanism. Such mechanism is typical for transglycosylases operating with retention of the anomeric configuration of the formed glycosidic bond and involving the formation of a covalent glycosyl-enzyme reaction intermediate.

General physiology and biophysics, 1998

Xyloglucan-endotransglycosylase (XET) is an enzyme involved in the metabolism of xyloglucan (XG) ... more Xyloglucan-endotransglycosylase (XET) is an enzyme involved in the metabolism of xyloglucan (XG) in plant cell walls and seeds. This enzyme acts both as a hydrolase and as a transglycosylase by transferring the fragments of xyloglucan molecules to other XG molecules or xyloglucan-derived oligosaccharides (XGOs). In this work, we studied the kinetics of interaction between XET and XG. The equilibrium in the reaction of XG degradation by XET was found to depend on the initial enzyme concentration and the availability of suitable glycosyl acceptors. After reaching the equilibrium, the addition to the reaction mixture of XET or XGOs caused further degradation of XG, and a new equilibrium with a higher degree of XG depolymerization was established. These results indicated that in the course of XG depolymerization, the enzyme is bound in a relatively stable, temporarily inactive enzyme-glycosyl complex and this complex is decomposed by transferring its glycosyl moiety to suitable oligosac...

Journal of Biological Chemistry, 2012

Background: Microarrays of plant-derived oligosaccharides are potentially powerful tools for the ... more Background: Microarrays of plant-derived oligosaccharides are potentially powerful tools for the high throughput discovery and screening of antibodies, enzymes, and carbohydrate-binding proteins. Results: Oligosaccharide microarrays were produced, and their utility was demonstrated in several applications. Conclusion: A new generation of oligosaccharide microarrays will make an important contribution to plant glycomic research. Significance: High throughput screening technology enables the more effective production of carbohydrate active enzymes and molecular probes.

BMC Plant Biology, 2008

Background Molecular probes are required to detect cell wall polymers in-situ to aid understandin... more Background Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Results Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the c...

Glycoconjugate Journal, 2009

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleav... more Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the nonreducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophorelabelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouseear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homoand hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.

Hyperosmotic growth medium containing 1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation... more Hyperosmotic growth medium containing 1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h and thereafter the growth resumed at almost the original rate. Fluorescent patches on the inner surface of cell walls stained with Calcofluor white was observed. The patches gradually disappeared in buds formed in hyperosmotic medium. Freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with amorphous cell-wall material. The rate of incorporation of D-[U-14C]glucose into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in the composition of the cell walls from osmotically stressed yeast and those from the control cells was found. Microtubules disappeared and actin patches were present in both mother cell and bud. After 2 - 3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarize...

Description de milieux geloses contenant de l'hydroxyethylcellulose coloree par liaison coval... more Description de milieux geloses contenant de l'hydroxyethylcellulose coloree par liaison covalente ou du xylane egalement colore. La secretion, dans le milieu, de cellulase ou de xylanase, se traduit par des aureoles non colorees autour des colonies notamment de Trichoderma reesei

Journal of Bacteriology, 1969

2-Deoxy- d -glucose (2DG) acted as a competitive inhibitor of the synthesis of cell wall componen... more 2-Deoxy- d -glucose (2DG) acted as a competitive inhibitor of the synthesis of cell wall components in Saccharomyces cerevisiae protoplasts. The synthesis of fibrillar glucan cell wall component was inhibited at a glucose to 2DG ratio of 4:1 in the cultivating medium. The completion of the formation of cell wall by the synthesis of the amorphous mannan-protein cell wall component was inhibited at a glucose to 2DG ratio of about 20:1. The inhibition could be reversed by increasing the glucose to 2DG ratio in the nutrient medium. No incorporation of 2DG into fibrillar glucan cell wall component was observed.

Cellular Microbiology, 2016

The cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylati... more The cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin-glucan covalent cell wall cross-links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors.

Biochemical Journal, 2001

To map the preferred cleavage sites of xyloglucan endotransglycosylases (XETs; EC 2.4.1.207) alon... more To map the preferred cleavage sites of xyloglucan endotransglycosylases (XETs; EC 2.4.1.207) along the donor substrate chain, we incubated the enzymes with tamarind (Tamarindus indica) xyloglucan (donor substrate; ≈ 205kDa; 21µM) plus the nonasaccharide [3H]XLLGol (Gal2·Xyl3·Glc3· [3H]glucitol; acceptor substrate; 0.6µM). After short incubation times, to minimize multiple cleavages, the size of the 3H-labelled transglycosylation products (determined by gel-permeation chromatography) indicated the positions of the cleavage sites relative to the non-reducing terminus of the donor. There was very little difference between the size profiles of the products formed by any of ten XETs tested [one native XET purified from cauliflower (Brassica oleracea) florets, four native XET isoenzymes purified from etiolated mung-bean (Phaseolus aureus) shoots, native XETs purified from lentil (Lens culinaris) and nasturtium (Tropaeolum majus) seeds, and three insect-cell-produced thale-cress (Arabidops...

FEMS yeast research, 2015

Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 fami... more Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 family of β(1,3)-glucanosyltransferases is essential for the determination of cell shape, for cell integrity and for virulence in pathogenic fungi. Candida albicans has five GH72 genes: PHR1 and PHR2 are pH dependent, the first being expressed at pH ≥ 6 and repressed at lower pH and the second regulated in the opposite manner, PGA4 is transcribed independently of pH whereas PHR3 and PGA5 have low expression levels. To characterize the catalytic properties of Phr1p-2p and probe the activity of Pga4p, we heterologously expressed these proteins and used a fluorescent assay based on the transfer of oligosaccharyl units from a donor to a sulforhodamine-labeled acceptor. Phr1p-2p used exclusively β-1,3-glucan or cell wall glucan as donor and laminarin-derived oligosaccharides as acceptor. The acceptor efficiency increased with the length of the oligosaccharide. The temperature optimum was 30°C. Th...

Plant Physiology and Biochemistry, 2001

Incubation of isolated NaCl-washed cell walls from epicotyls of pea (Pisum sativum) and nasturtiu... more Incubation of isolated NaCl-washed cell walls from epicotyls of pea (Pisum sativum) and nasturtium (Tropaeolum majus) with solutions of various oligosaccharides released among others the cell wall marker enzyme xyloglucan endotransglycosylase (XET, EC 2.4.1.207). The greatest release of XET occurred upon incubation of the cell walls with xyloglucanderived oligosaccharides (XGOS, DP 7-9). Concomitantly, reduced radioactive nonasaccharide [ 3 H]-XLLGol (Gal 2 .Xyl 3 .Glc 3 .[1-3 H]-glucitol) was incorporated into the cell walls. Subsequent hydrolysis of the radioactively labelled cell walls with Trichoderma cellulase liberated XGOS-alditols, DP 7-9 as the sole radioactive products indicating that [ 3 H]-XLLGol was incorporated into the cell wall xyloglucan by transglycosylation, as an entity. Oligosaccharides of cello-, chito-and/or oligoglucurono-series were much less effective than XGOS but a substantial liberation of XET and other proteins from plant cell walls could be achieved by the nucleophile 0.1 M imidazole. The specific release of the cell wall-associated XET activity by incubation with xyloglucan-derived oligosaccharides and the simultaneous incorporation of the tritiated xyloglucan nonasaccharide en bloc into the cell walls indicates that XET is present in the cell walls in form of a competent glycosyl-enzyme complex which decomposes by transglycosylation of its glycan moiety to added xyloglucan-oligosaccharide acceptors. This finding suggests a new concept for the regulation of activity of cell wall-associated glycanases/transglycosylases: they exist in plant cell walls in a transiently latent state as covalent glycosyl-enzyme complexes and are active only when suitable glycosyl acceptors become available. © 2001 Éditions scientifiques et médicales Elsevier SAS cell walls / nasturtium / Pisum sativum / transglycosylation / Tropaeolum majus / XET / xyloglucan / xyloglucanendotransglycosylase XET, xyloglucan endotransglycosylase (EC 2.4.1.207) / XG, xyloglucan / XGOS, xyloglucan-derived oligosaccharides

Journal of General Microbiology, 1990

Exposure of dark-grown mycelia of Trichodevma tliride to white light elicited a transient burst o... more Exposure of dark-grown mycelia of Trichodevma tliride to white light elicited a transient burst of respiratory activity manifested as increased O2 consumption which was not paralleled by a corresponding increase in C 0 2 production. The period of increased uptake of O2 lasted for 5-10min and was independent of the duration of illumination. The inhibitors of respiration tested, antimycin A and mucidin, and the antioxidant, dithiothreitol, effectively suppressed the photostimulated uptake of 02, whereas rotenone, amytal and salicylhydroxamic acid were without effect. It is concluded that the illumination of mycelia caused irreversible photo-oxidation of an as yet unidentified compound, and that the electrons released by the photochemical event were accepted by a NADindependent flavin dehydrogenase and further transferred to atmospheric O2 via the cytochrome electrontransport chain coupled with the formation of ATP.

Acta Biotechnologica, 1987

Intracellular concentrations of ATP, cyclic AMP and glucose‐6‐phosphate were monitored during gro... more Intracellular concentrations of ATP, cyclic AMP and glucose‐6‐phosphate were monitored during growth of partially catabolically derepressed strain of Trichoderma reesei CC II in medium containing lactose as the sole carbon source. The induction of cellulase synthesis occured when the concentration of lactose in the medium decreased below 7 mg/ml. The onset of cellulase synthesis was preceded by a transient peak of intracellular concentration of ATP and by the increase of the cyclic AMP contents in the mycelium whereby the intracellular level of glucose‐6‐phosphate was at its minimum. By keeping the lactose concentration in the medium at 2 mg/ml, it was possible to support the continuation of cellulase synthesis over the prolonged periods without appreciable growth of biomass.

The FEBS Journal, 2015

Covalent cross‐links between chitin and glucan at the yeast cell wall are created by the transgly... more Covalent cross‐links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β‐1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three‐dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3‐1,4‐β‐d‐glucanase, followed by site‐directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β‐1,3‐glucan, β‐1,6‐glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nu...

European Journal of Biochemistry, 2005

Soluble and membrane-bound phosphatase and phosphodiesterase activities are present in preparatio... more Soluble and membrane-bound phosphatase and phosphodiesterase activities are present in preparations of 1,3-j-~-glucan synthase from pea epicotyls. UDP-glucose phosphodiesterase and non-specific alkaline phosphatase could be partially inhibited by N-ethylmaleimide or iodoacetamide and partially removed from membranes by washing. Such treatments helped to prolong 1,3-fl-glucan synthase activity. Nevertheless, the 1,3-fl-~-glucan synthase activity in washed membranes still gradually decreased during incubation in buffer at 30°C. The rate of decay was reduced by adding more specific phosphatase inhibitors, e. g. molybdate, vanadate or fluoride, or by addition of nucleotides, and much of the loss of 1,3-/3-~-glucan synthase activity during preincubation could be restored by addition of phosphatidylethanolamine to the assay mixtures. It is concluded that membrane phospholipid is an essential part of the environment of 1,3-P-glucan synthase and must be maintained intact in order for the enzyme to remain fully active.

Plant Physiology and Biochemistry, 2003

Two isoenzymes of xyloglucan endo-transglycosylase (XET, EC 2.4.1.207) were identified in nasturt... more Two isoenzymes of xyloglucan endo-transglycosylase (XET, EC 2.4.1.207) were identified in nasturtium (Tropaeolum majus L.), so far. One is located in seeds (sXET) and is expressed during germination. The other enzyme (eXET) is confined to epicotyls and other growing regions. In this work, we examined catalytic properties of the two XETs and tried to find a correlation with their presumed

Plant Physiology and Biochemistry, 2010

Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric poin... more Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric points (pI) were detected in crude extracts from germinating nasturtium seeds. Without further fractionation, all five forms behaved as typical endotransglycosylases since they exhibited only transglycosylating (XET) activity and no xyloglucan-hydrolysing (XEH) activity. They all were glycoproteins with identical molecular mass, and deglycosylation led to a decrease in molecular mass from approximately 29 to 26.5 kDa. The major enzyme form having pI 6.3, temporarily designated as TmXET(6.3), was isolated and characterized. Molecular and biochemical properties of TmXET(6.3) confirmed its distinction from the XTHs described previously from nasturtium. The enzyme exhibited broad substrate specificity by transferring xyloglucan or hydroxyethylcellulose fragments not only to oligoxyloglucosides and cellooligosaccharides but also to oligosaccharides derived from b-(1,4)-D-glucuronoxylan, b-(1,6)-D-glucan, mixed-linkage b-(1,3; 1,4)-D-glucan and at a relatively low rate also to b-(1,3)-gluco-oligosaccharides. The transglycosylating activity with xyloglucan as donor and cello-oligosaccharides as acceptors represented 4.6%, with laminarioligosaccharides 0.23%, with mixed-linkage b-(1,3; 1,4)-D-gluco-oligosaccharides 2.06%, with b-(1,4)-D-glucuronoxylo-oligosaccharides 0.31% and with b-(1,6)-D-gluco-oligosaccharides 0.69% of that determined with xyloglucan oligosaccharides as acceptors. Based on the sequence homology of tryptic fragments with the sequences of known XTHs, the TmXET(6.3) was classified into group II of the XTH phylogeny of glycoside hydrolase family GH16.