Institute for Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University, Stefan-Meier-Straße 19, 79104 Freiburg, Germany.
Institute of Organic Chemistry, Albert-Ludwigs-Universität, Albertstrasse 21, 79104 Freiburg, Germany.
Molecules. 2020 Jul 27;25(15):3400. doi: 10.3390/molecules25153400.
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 - and -glycosyltransferase from NRRL2338. When feeding experiments with 1,4-diaminoanthraquinone in 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, -α-glucosyl-1,4-diaminoanthraquinone and -β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a ,-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 J1074, containing the dTDP-d-glucose synthase gene and the plasmid pUWL-A-, was used as host, U3 was converted to the same compounds. Protein production in and purification of Sace_3599 was carried out. The enzyme showed glycosyl hydrolase activity and was able to produce mono- and di--glycosylated products in vitro. When UDP-α-d-glucose was used as a sugar donor, U3 was stereoselective converted to -β-glucosyl-1,4-diaminoanthraquinone and ,-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 - and -glycosidic bonds.
糖基转移酶是一类重要的酶，常用于生成新型天然产物。在本研究中，我们描述了从 NRRL2338 中鉴定和表征的一种反转及糖基转移酶。当在 中进行 1,4-二氨基蒽醌的喂养实验时，通过 HPLC-MS 观察到新化合物 (U3G 和 U3DG) 的形成。通过 NMR 进行的结构阐明表明，U3G 由两种化合物组成，即 -α-葡萄糖基-1,4-二氨基蒽醌和 -β-葡萄糖基-1,4-二氨基蒽醌。根据 UV 和 MS 数据，U3DG 是一种,-二葡萄糖基-1,4-二氨基蒽醌。为了找到负责的糖基转移酶，进行了基因缺失实验，我们鉴定了属于 CAZy 家族 1 的糖基转移酶 Sace_3599。当使用含有 dTDP-d-葡萄糖合酶基因 和质粒 pUWL-A- 的 J1074 作为宿主时，U3 被转化为相同的化合物。在 和 Sace_3599 的蛋白生产和纯化进行。该酶表现出糖苷水解酶活性，并能够在体外产生单糖和二糖产物。当 UDP-α-d-葡萄糖作为糖供体时，U3 被立体选择性地转化为 -β-葡萄糖基-1,4-二氨基蒽醌和,-二葡萄糖基-1,4-二氨基蒽醌。在体外实验中使用 1,4-二羟基蒽醌作为底物也导致形成单糖基化和二糖基化产物，但产率较低。总的来说，我们鉴定和表征了一种新型的糖基转移酶，它具有糖基水解酶活性和糖基化“药物样”结构的能力，形成 - 和 - 糖苷键。
