Synthese funktionalisierter Zuckernukleotid-Donoren für die Microarray-basierte Identifizierung und Charakterisierung pflanzlicher Glykosyltransferasen
Biochemie und Biophysik der Pflanzen
Zusammenfassung der Projektergebnisse
Plant cell walls consist of a sophisticated composite largely made of several polysaccharide networks with essential functions in the life cycle of the plant. Cell wall polysaccharides are among the most complex glycans in nature and differ in the structure and amount of the monosaccharides, the type of connections between them, and other modifications. These polysaccharides receive an enormous interest as sources of sustainable materials and for the production of biofuels. The generation of genetically modified plants with optimized cell wall composition may be a promising strategy to enhance the economic value of biomass as a renewable resource. Properties of the plant cell wall such as growth, biomass recalcitrance, and material strengths are determined by plant cell wall biosynthetic genes. Although knowledge of the glycosyltransferases (GTs) that assemble the highly complex and heterogeneous glycans constructing the cell wall is critical for controlling their biosynthesis and thus for tailoring the plant’s properties, our knowledge of the functions of plant cell wall GTs is still very limited. We recently produced a glycan microarray equipped with synthetic cell wall oligosaccharides. These oligosaccharides represent fragments of natural hemicellulose and pectin polysaccharides, including arabinoxylan-, type I and type II arabinogalactan-, xyloglucan-, and mixed-linkage glucan-related structures. This microarray provided for the first time the opportunity to develop an assay for the simultaneous screening of various plant glycosyltransferases with a large number of acceptor substrates. When the microarray is incubated with azido-functionalized sugar nucleotide donors and putative glycosyltransferases, any incorporated monosaccharide can be visualized by subsequent labeling with a fluorescent dye using click-chemistry. Thus, it is possible to identify any active combination of nucleotide sugar donor, acceptor substrate, and glycosyltransferase, providing the first high-throughput screening platform for plant glycosyltransferases. We have chemically synthesized or commercially procured different azido-functionalized sugar nucleotides based on galactose, fucose, xylose, rhamnose, and arabinofuranose. These nucleotides were prepared by coupling of the respective stereochemically defined sugar 1-phosphates with activated uridine monophosphate (UMP). We then expressed a number of the catalytic extracellular domains of putative plant glycosyltransferases in human embryonic kidney cells (HEK) 293 and investigated these using the newly developed glycan microarray assay. Reported glycosyltransferase activities were corrected or defined more precisely and a new galactosyltransferase was identified. Through additional experiments using high pressure liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy, we found that this galactosyltransferase adds galactose in a β-1,3-linkage to β-1,6-linked galactan-oligosaccharides, a reactivity that has not been reported so far. Currently prepared mutant plants either overexpressing or lacking this galactosyltransferase followed by analysis of their phenotypes and cell wall polysaccharide composition will provide further inside into the biological function of this newly identified glycosyltransferase. Other enzymes could not be expressed in a functional form or did not accept the unnatural modification in the sugar nucleotide donor. In the latter cases, HPLC-experiments were performed to analyze their activities, and information on the substrate specificities of a rhamnosyl- and an arabinosyltransferase were obtained. The opportunity to express and screen large numbers of glycosyltransferases instead of rationally selected candidates will markedly accelerate the elucidation of plant cell wall biosynthetic pathways. Advances in plant cell wall biosynthesis research will set the stage for production of tailor-made plants with improved properties, including crop resistance to pathogens, biomass digestibility, material strength, and the shelf life of fruits and vegetables.
Projektbezogene Publikationen (Auswahl)
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Chemo‐Enzymatic Synthesis of Long‐Chain Oligosaccharides for Studying Xylan‐Modifying Enzymes. Chemistry – A European Journal, 29(26).
Álvarez‐Martínez, Ignacio; Ruprecht, Colin; Senf, Deborah; Wang, Hsin‐tzu; Urbanowicz, Breeanna R. & Pfrengle, Fabian
