Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i>
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...
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MDPI AG
2020-07-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/15/3400 |
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| author | Fabienne Gutacker Yvonne-Isolde Schmidt-Bohli Tina Strobel Danye Qiu Henning Jessen Thomas Paululat Andreas Bechthold |
| author_facet | Fabienne Gutacker Yvonne-Isolde Schmidt-Bohli Tina Strobel Danye Qiu Henning Jessen Thomas Paululat Andreas Bechthold |
| author_sort | Fabienne Gutacker |
| collection | DOAJ |
| description | 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><sub>1</sub>-α-glucosyl-1,4-diaminoanthraquinone and <i>N</i><sub>1</sub>-β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a <i>N</i><sub>1</sub>,<i>N</i><sub>4</sub>-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-<span style="font-variant: small-caps;">d</span>-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 in vitro. When UDP-α-<span style="font-variant: small-caps;">d</span>-glucose was used as a sugar donor, U3 was stereoselective converted to <i>N</i><sub>1</sub>-β-glucosyl-1,4-diaminoanthraquinone and <i>N</i><sub>1</sub>,<i>N</i><sub>4</sub>-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 <i>N</i>- and <i>O</i>-glycosidic bonds. |
| first_indexed | 2024-03-10T18:10:53Z |
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| language | English |
| last_indexed | 2024-03-10T18:10:53Z |
| publishDate | 2020-07-01 |
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| spelling | doaj.art-32cf25f9d60e4cc28971627ad7e4acc32023-11-20T08:07:05ZengMDPI AGMolecules1420-30492020-07-012515340010.3390/molecules25153400Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i>Fabienne Gutacker0Yvonne-Isolde Schmidt-Bohli1Tina Strobel2Danye Qiu3Henning Jessen4Thomas Paululat5Andreas Bechthold6Institute for Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University, Stefan-Meier-Straße 19, 79104 Freiburg, GermanyInstitute for Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University, Stefan-Meier-Straße 19, 79104 Freiburg, GermanyInstitute for Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University, Stefan-Meier-Straße 19, 79104 Freiburg, GermanyInstitute of Organic Chemistry, Albert-Ludwigs-Universität, Albertstrasse 21, 79104 Freiburg, GermanyInstitute of Organic Chemistry, Albert-Ludwigs-Universität, Albertstrasse 21, 79104 Freiburg, GermanyOrganic Chemsitry II, Universität Siegen, Adolf-Reichwein-Strasse 2, 57068 Siegen, GermanyInstitute for Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University, Stefan-Meier-Straße 19, 79104 Freiburg, GermanyGlycosyltransferases 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><sub>1</sub>-α-glucosyl-1,4-diaminoanthraquinone and <i>N</i><sub>1</sub>-β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a <i>N</i><sub>1</sub>,<i>N</i><sub>4</sub>-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-<span style="font-variant: small-caps;">d</span>-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 in vitro. When UDP-α-<span style="font-variant: small-caps;">d</span>-glucose was used as a sugar donor, U3 was stereoselective converted to <i>N</i><sub>1</sub>-β-glucosyl-1,4-diaminoanthraquinone and <i>N</i><sub>1</sub>,<i>N</i><sub>4</sub>-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 <i>N</i>- and <i>O</i>-glycosidic bonds.https://www.mdpi.com/1420-3049/25/15/3400glycosyltransferaseglycohydrolase<i>Saccharopolyspora erythraea</i>anthraquinoneglycobiologynucleotide-activated sugar donor |
| spellingShingle | Fabienne Gutacker Yvonne-Isolde Schmidt-Bohli Tina Strobel Danye Qiu Henning Jessen Thomas Paululat Andreas Bechthold Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i> Molecules glycosyltransferase glycohydrolase <i>Saccharopolyspora erythraea</i> anthraquinone glycobiology nucleotide-activated sugar donor |
| title | Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i> |
| title_full | Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i> |
| title_fullStr | Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i> |
| title_full_unstemmed | Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i> |
| title_short | Identification and Characterization of a Novel <i>N</i>- and <i>O</i>-Glycosyltransferase from <i>Saccharopolyspora erythraea</i> |
| title_sort | identification and characterization of a novel i n i and i o i glycosyltransferase from i saccharopolyspora erythraea i |
| topic | glycosyltransferase glycohydrolase <i>Saccharopolyspora erythraea</i> anthraquinone glycobiology nucleotide-activated sugar donor |
| url | https://www.mdpi.com/1420-3049/25/15/3400 |
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