Identification and Characterization of a Novel N- and O-Glycosyltransferase from Saccharopolyspora erythraea

Glycosyltransferases are important enzymes which are often used as tools to generate novel natural products. In this study, we describe the identification and characterization of an inverting N- and O-glycosyltransferase from Saccharopolyspora erythraea NRRL2338. When feeding experiments with 1,4-diaminoanthraquinone in Saccharopolyspora erythraea were performed, the formation of new compounds (U3G and U3DG) was observed by HPLC-MS. Structure elucidation by NMR revealed that U3G consists of two compounds, N(1)-α-glucosyl-1,4-diaminoanthraquinone and N(1)-β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a N(1),N(4)-diglucosyl-1,4-diaminoanthraquinone. In order to find the responsible glycosyltransferase, gene deletion experiments were performed and we identified the glycosyltransferase Sace_3599, which belongs to the CAZy family 1. When Streptomyces albus J1074, containing the dTDP-d-glucose synthase gene oleS and the plasmid pUWL-A-sace_3599, was used as host, U3 was converted to the same compounds. Protein production in Escherichia coli and purification of Sace_3599 was carried out. The enzyme showed glycosyl hydrolase activity and was able to produce mono- and di-N-glycosylated products in vitro. When UDP-α-d-glucose was used as a sugar donor, U3 was stereoselective converted to N(1)-β-glucosyl-1,4-diaminoanthraquinone and N(1),N(4)-diglucosyl-1,4-diaminoanthraquinone. The use of 1,4-dihydroxyanthraquinone as a substrate in in vitro experiments also led to the formation of mono-glucosylated and di-glucosylated products, but in lower amounts. Overall, we identified and characterized a novel glycosyltransferase which shows glycohydrolase activity and the ability to glycosylate “drug like” structures forming N- and O-glycosidic bonds.