Pyranose Dehydrogenase from litter-decomposing fungi

The presented project shall deliver the first molecular biological information about a novel enzyme, make the protein available for detailed studies pertaining to its structure and function as well as to technological applications, and elucidate the biological function of this and related enzymes. Oxidoreductases of free sugars are typical of a large number of higher fungi. Pyranose dehydrogenase (PDH) was recently isolated from Agaricus bisporus, it catalyzes the oxidation of various mono- and oligosaccharides at position C-2 and/or C-3. It is characterized in only a small number of fungi, no gene encoding a PDH has been cloned thus far. Various functions in the biodegradation of lignocellulose are being discussed for related enzymes such as CDH and P2O, but are to date still largely speculative. Enzymatic sugar transformations present a great potential for the synthesis of, e.g., various keto sugars. By combining enzymatic and chemical reactions, a vast number of sugar-derived compounds can be constructed. PDH can be a valuable tool for such applications. "Litter-decomposing" fungi, the only known source of these enzymes, grow slowly in cultivation, preparation of even minor amounts of enzyme takes several weeks. Therefore, cloning of the gene and production of the enzyme by heterologous expression is a prerequisite for both application and more detailed research. We plan to clone and characterize PDH-encoding genes from selected sources; cDNA clones will be isolated for expression in P. pastoris, and the protein will be characterized especially regarding electron acceptors, structural requirements of electron donors and reaction products (cooperation with Prof. J. Volc, Czech Academy of Sciences). The crystal structure of PDH shall be determined (cooperation with Dr. C. Divne, Royal Institute of Technology Stockholm), and construction of a PDH-negative strain of Agaricus and characterization of the resulting phenotype will elucidate the biological function of PDH, together with ultrastructural investigations of PDH-forming and deficient strains during growth on lignocellulose substrates (cooperation with Prof. G. Daniel (Swedish University of Agricultural Sciences, Uppsala).

The presented project shall deliver the first molecular biological information about a novel enzyme, make the protein available for detailed studies pertaining to its structure and function as well as to technological applications, and elucidate the biological function of this and related enzymes. Oxidoreductases of free sugars are typical of a large number of higher fungi. Pyranose dehydrogenase (PDH) was recently isolated from Agaricus bisporus, it catalyzes the oxidation of various mono- and oligosaccharides at position C-2 and/or C-3. It is characterized in only a small number of fungi, no gene encoding a PDH has been cloned thus far. Various functions in the biodegradation of lignocellulose are being discussed for related enzymes such as CDH and P2O, but are to date still largely speculative. Enzymatic sugar transformations present a great potential for the synthesis of, e.g., various keto sugars. By combining enzymatic and chemical reactions, a vast number of sugar-derived compounds can be constructed. PDH can be a valuable tool for such applications. "Litter-decomposing" fungi, the only known source of these enzymes, grow slowly in cultivation, preparation of even minor amounts of enzyme takes several weeks. Therefore, cloning of the gene and production of the enzyme by heterologous expression is a prerequisite for both application and more detailed research. We plan to clone and characterize PDH-encoding genes from selected sources; cDNA clones will be isolated for expression in P. pastoris, and the protein will be characterized especially regarding electron acceptors, structural requirements of electron donors and reaction products (cooperation with Prof. J. Volc, Czech Academy of Sciences). The crystal structure of PDH shall be determined (cooperation with Dr. C. Divne, Royal Institute of Technology Stockholm), and construction of a PDH-negative strain of Agaricus and characterization of the resulting phenotype will elucidate the biological function of PDH, together with ultrastructural investigations of PDH-forming and deficient strains during growth on lignocellulose substrates (cooperation with Prof. G. Daniel (Swedish University of Agricultural Sciences, Uppsala).