Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i>

• Main Authors: Fabienne Gutacker, Yvonne-Isolde Schmidt-Bohli, Tina Strobel, Danye Qiu, Henning Jessen, Thomas Paululat, Andreas Bechthold
• Format: Article
• Language: English
• Published: MDPI AG 2020-07-01
• Series: Molecules
• Subjects: glycosyltransferase; glycohydrolase; <i>Saccharopolyspora erythraea</i>; anthraquinone; glycobiology; nucleotide-activated sugar donor
• Online Access: https://www.mdpi.com/1420-3049/25/15/3400

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 <i>N</i>- and <i>O</i>-glycosyltransferase from <i>Saccharopolyspora erythraea</i> NRRL2338. When feeding experiments with 1,4-diaminoanthraquinone in <i>Saccharopolyspora erythraea </i>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, <i>N</i>₁-α-glucosyl-1,4-diaminoanthraquinone and <i>N</i>₁-β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a <i>N</i>₁,<i>N</i>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 <i>Streptomyces albus</i> J1074, containing the dTDP-d-glucose synthase gene <i>oleS</i> and the plasmid pUWL-A-<i>sace_3599</i>, was used as host, U3 was converted to the same compounds. Protein production in <i>Escherichia coli</i> and purification of Sace_3599 was carried out. The enzyme showed glycosyl hydrolase activity and was able to produce mono- and di-<i>N</i>‑glycosylated products <i>in vitro</i>. When UDP-α-d-glucose was used as a sugar donor, U3 was stereoselective converted to <i>N</i>₁-β-glucosyl-1,4-diaminoanthraquinone and <i>N</i>₁,<i>N</i>₄-diglucosyl-1,4-diaminoanthraquinone. The use of 1,4-dihydroxyanthraquinone as a substrate in <i>in vitro</i> 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 <i>N</i>- and <i>O</i>-glycosidic bonds.