Chitin exists in yeast cells both as free and bound in a complex with β-1,3/β-1,6-glucan. The formation of covalent links between chitin and β-glucans is catalyzed by the enzymes Crh1 and Crh2, acting as transglycosylases. We found that N-acetyl-chito-oligosaccharides, as well as laminarioligosaccharides, the respective products of partial hydrolysis of chitin, and β-1,3-glucan, interfered with reactions catalyzed by Crh1p and Crh2p in vitro. However, the N-acetyl-chito-oligosaccharides did not influence the growth rate of the yeast, neither did they affect the yeast phenotype, but they prolonged the lag phase. Inhibition of Crh1 and Crh2 in vivo with oligosaccharides derived from chitin leads to an increase of alkali-soluble chitin and a decrease in the amount of chitin linked to β-glucans. In addition, yeast cells growing in the presence of N-acetyl-D-chito-oligosaccharides accumulated more chitin than control cells.
KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-β-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.
- MeSH
- fylogeneze MeSH
- glykosylace MeSH
- glykosyltransferasy chemie metabolismus MeSH
- klíčení MeSH
- komplementární DNA genetika MeSH
- molekulární modely MeSH
- petržel (rod) enzymologie MeSH
- proteinové inženýrství * MeSH
- rostlinné proteiny chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- semena rostlinná enzymologie MeSH
- strukturní homologie proteinů MeSH
- substrátová specifita MeSH
- Tropaeolum enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- biochemie MeSH
- mikrobiologie MeSH
- Publikační typ
- biografie MeSH
- Geografické názvy
- Slovenská republika MeSH
- O autorovi
- Šubík, Július, 1944- Autorita
- MeSH
- buněčná stěna chemie účinky léků MeSH
- elektronová mikroskopie metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- hydrolasy chemie škodlivé účinky MeSH
- osmotický tlak fyziologie MeSH
- osmóza fyziologie účinky záření MeSH
- proteasy chemie škodlivé účinky MeSH
- soli chemie škodlivé účinky MeSH
- Zygosaccharomyces cytologie chemie MeSH
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- finanční podpora výzkumu jako téma MeSH
- glykosyltransferasy izolace a purifikace metabolismus MeSH
- hydrolasy izolace a purifikace metabolismus MeSH
- isoelektrická fokusace metody MeSH
- izoenzymy izolace a purifikace metabolismus MeSH
- rostliny enzymologie MeSH
