Heteronuclear NMR studies of 13C-labeled yeast cell wall ?-glucan oligosaccharides

Yu, Liping; Goldman, Robert C.; Sullivan, Patrick A.; Walker, Greg F.; Fesik, Stephen W.

doi:10.1007/bf00176009

Cited by 42 publications

(27 citation statements)

References 32 publications

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“…Both enzymes cleave a ␤-(1-3)-glucan chain and transfer the nonreducing end portion to the nonreducing end of another ␤-(1-3)-glucan chain. The first type (BGT1) specifically hydrolyzes a laminaribiose unit from the reducing end of a ␤-(1-3)-glucan chain and then transfers the remainder to the nonreducing end of another ␤-(1-3)-glucan chain, forming a new intrachain ␤-(1-6)-linkage during transfer (16,17,19). This transferase has been found in C. albicans (16) and in S. cerevisiae and both are encoded by the BGL2 gene (19).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a secreted ␤-(1-3)-glucanosyltransferase (referred to as BGT1 in this paper) was isolated from C. albicans, which catalyzes the transfer of a segment of ␤-(1-3)-glucan to the nonreducing end of another ␤-(1-3)-glucan chain, forming a new, intrachain ␤-(1-6)-linkage in the process (16,17). The enzyme's function in the wall is unknown since disruption of the BGL2 gene encoding the homolog in S. cerevisiae did not show a distinct phenotype (18,19).…”

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confidence: 99%

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A Novel β-( , , )-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus

Hartland¹,

Fontaine²,

Debeaupuis³

et al. 1996

Journal of Biological Chemistry

112

103

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A Novel β-( , , )-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus

Hartland¹,

Fontaine²,

Debeaupuis³

et al. 1996

Journal of Biological Chemistry

112

103

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“…An exoglucanase, purified from C. albicans [16], catalyzed a glucosyltransferase reaction in the presense of appropriate concentrations of acceptor molecules, yielding linear ,8-1,3 glucan oligomers. In addition, a novel glucosyltransferase was purified from C. albicans [12], which catalyzed a novel transferase reac- tion yielding a linear glucan [13] in which the donor addition occurred as a p-1,6 linkage at the C6 site on the nonreducing end of the acceptor glucan chain [13]. We developed a sensitive assay using radiolabeled substrates and HPLC analysis of reaction products to characterize the enzyme activity purified from S. cerevisiae.…”

Section: Discussionmentioning

confidence: 99%

“…Preliminary NMR analysis revealed the presence of a p-1,6 linkage in the product glucan, and the product structure was believed to be a branched glucan. However, the product was still a linear glucan, but one in which the donor addition occurred as a p-1,6 linkage at the C6 site on the nonreducing end of the acceptor glucan chain, as revealed by more detailed NMR analysis [13]. Thus, two initially separate /?-1,3 glucan chains were joined by connecting the reducing terminus of the activated donor chain to the nonreducing terminus of the acceptor chain in a p-1,6 linkage, yielding a 'kinked', but still linear glucan chain.…”

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confidence: 92%

Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene

Goldman

Sullivan

Zakula

et al. 1995

European Journal of Biochemistry

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Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor p-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor /3-1,3 glucan, yielding a linear glucan containing a p-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429-4411, An apparent K,, of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent K, of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H,O competing for reaction at the activated donor site, thus resulting in hydrolysis.However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for p-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of p-1,3 glucan into the previously existing cell wall structure.Keywords. Glucosyltransferase ; fungal wall ; BGL2 protein ; wall assembly ; Saccharomyces.Synthesis and assembly of the fungal cell wall is a complex process about which we know much less compared to the analogous events occurring in bacteria. We do know that linear polymers of chitin, p-1,4 N-acetylglucosamine, and glucan, p-1,3 glucose, are synthesized by microsomal fractions prepared from fungal cells when iincubated in the presence of their appropriate UDP-sugars [l, 21. The process appears to be vectorial in that UDP-sugar presenf in the cytoplasm is polymerized, with concurrent translocation through the membrane of the growing polymeric chain. However, little if anything is known about the following aspects of p-1,3 polymer synthesis: (a) initiation of chain synthesis, (b) regulation of chain initiation, (c) translocation through the membrane, (d) mechanism and regulation of termination of chain synthesis, and (e) localization of chain synthesis during different phases of wall expansion. Furthermore, glucan found in the mature cell wall consists of highly branched molecules containing both p-1,3 and p-1,6 linkages.

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“…These could be assigned by their vicinal couplings in an HH-COSY spectrum. [29][30][31][32]. The Lorentzian tails associated with the rather high linewidth of the 1H-NMR resonances (approximately 7 Hz) combined with the low dispersion of the chemical shift precluded an accurate estimate of impurities from the proton and HH-COSY NMR spectra alone.…”

Section: Product Characterizationmentioning

confidence: 99%

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

Antelo¹,

Cosio

Hertkorn

et al. 1998

FEBS Letters

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A (1 ~ 3)-~-glucan synthase activity was identified in cell membrane preparations from the oomycete Phytophthora sojae, a soybean pathogen. The activity could be solubilized using the zwitterionic detergent CHAPS at relatively low concentrations (3 mg/ml). High salt concentrations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increase of calculated Vma× values (2.5 vs. 0.4 nkat/mg protein) but only minor alteration of the Km (10.6 vs. 10.7 raM). Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular weight (1 ~ 3)-[~-glucan. Glucan synthase activity in both membrane and solubilized preparations was not activated by GTP or divalent cations as reported for other fungal or plant glucan synthases, The activity was inhibited, as expected, in a competitive manner by UDP with a Ki of 2.9 raM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment. This procedure resulted in the selective enrichment of a protein band with apparent Mr 108 000 in SDS-PAGE which was not visible in any of the steps preceding product entrapment. The glucan pellets from product entrapment contained up to 3% of the initial enzyme activity present in the fraction used for the procedure.

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Heteronuclear NMR studies of 13C-labeled yeast cell wall ?-glucan oligosaccharides

Cited by 42 publications

References 32 publications

A Novel β-( , , )-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus

A Novel β-( , , )-Glucanosyltransferase from the Cell Wall of Aspergillus fumigatus

Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

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