Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5
[Display omitted] ► Fluorescent lipid diphosphate sugar. ► WbwC and WbdN substrate. ► Fluorescent assays. The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural...
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| Veröffentlicht in: | Carbohydrate research 2013-01, Vol.366 (25), p.17-24 |
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| creator | Vinnikova, Anna N. Druzhinina, Tatyana N. Danilov, Leonid L. Utkina, Natalia S. Torgov, Vladimir I. Veselovsky, Vladimir V. Wang, Shuo Liu, Bin Wang, Lei Brockhausen, Inka |
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► Fluorescent lipid diphosphate sugar. ► WbwC and WbdN substrate. ► Fluorescent assays.
The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-d-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars. |
| doi_str_mv | 10.1016/j.carres.2012.11.009 |
| format | Article |
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► Fluorescent lipid diphosphate sugar. ► WbwC and WbdN substrate. ► Fluorescent assays.
The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-d-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars.</description><identifier>ISSN: 0008-6215</identifier><identifier>EISSN: 1873-426X</identifier><identifier>DOI: 10.1016/j.carres.2012.11.009</identifier><identifier>PMID: 23261778</identifier><language>eng</language><publisher>Netherlands: Elsevier Ltd</publisher><subject>Biocatalysis ; Chemical synthesis ; E. coli O157 ; E. coli O5 ; Enterohemorrhagic Escherichia coli - enzymology ; Enterohemorrhagic Escherichia coli - metabolism ; Escherichia coli O157 ; Escherichia coli O157 - enzymology ; Escherichia coli O157 - metabolism ; fluorescence ; Fluorescent assay ; Fluorescent Dyes - chemistry ; Fluorescent Dyes - metabolism ; genes ; Glycosyltransferases ; Glycosyltransferases - metabolism ; Gram-negative bacteria ; Molecular Structure ; O Antigens - biosynthesis ; O Antigens - chemistry ; Stereoisomerism ; sugars ; WbdN</subject><ispartof>Carbohydrate research, 2013-01, Vol.366 (25), p.17-24</ispartof><rights>2012 Elsevier Ltd</rights><rights>Copyright © 2012 Elsevier Ltd. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-a10257f8dc9962cf56fdac2d4e78a9669190fbde1e4a0f437db64637544a9b783</citedby><cites>FETCH-LOGICAL-c386t-a10257f8dc9962cf56fdac2d4e78a9669190fbde1e4a0f437db64637544a9b783</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0008621512004661$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23261778$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vinnikova, Anna N.</creatorcontrib><creatorcontrib>Druzhinina, Tatyana N.</creatorcontrib><creatorcontrib>Danilov, Leonid L.</creatorcontrib><creatorcontrib>Utkina, Natalia S.</creatorcontrib><creatorcontrib>Torgov, Vladimir I.</creatorcontrib><creatorcontrib>Veselovsky, Vladimir V.</creatorcontrib><creatorcontrib>Wang, Shuo</creatorcontrib><creatorcontrib>Liu, Bin</creatorcontrib><creatorcontrib>Wang, Lei</creatorcontrib><creatorcontrib>Brockhausen, Inka</creatorcontrib><title>Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5</title><title>Carbohydrate research</title><addtitle>Carbohydr Res</addtitle><description>[Display omitted]
► Fluorescent lipid diphosphate sugar. ► WbwC and WbdN substrate. ► Fluorescent assays.
The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-d-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars.</description><subject>Biocatalysis</subject><subject>Chemical synthesis</subject><subject>E. coli O157</subject><subject>E. coli O5</subject><subject>Enterohemorrhagic Escherichia coli - enzymology</subject><subject>Enterohemorrhagic Escherichia coli - metabolism</subject><subject>Escherichia coli O157</subject><subject>Escherichia coli O157 - enzymology</subject><subject>Escherichia coli O157 - metabolism</subject><subject>fluorescence</subject><subject>Fluorescent assay</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Fluorescent Dyes - metabolism</subject><subject>genes</subject><subject>Glycosyltransferases</subject><subject>Glycosyltransferases - metabolism</subject><subject>Gram-negative bacteria</subject><subject>Molecular Structure</subject><subject>O Antigens - biosynthesis</subject><subject>O Antigens - chemistry</subject><subject>Stereoisomerism</subject><subject>sugars</subject><subject>WbdN</subject><issn>0008-6215</issn><issn>1873-426X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc9u1DAQhyMEokvhDRD4yCXBdhI7uSChqvyRKu2hVOJmOfZ441USL55kpTxNXxUvKRw52WP95htrvix7y2jBKBMfj4XRMQIWnDJeMFZQ2j7LdqyRZV5x8fN5tqOUNrngrL7KXiEeU0mFFC-zK15ywaRsdtnj_TrNPaBHEhzRxA1LSFAD00y0MXCaQyS4dDhHPQNxqToMqwm4DullQgdRIyDx0zkMZ7DpQhKPaEQYu2G9UPe5nmZ_gOnPjESGGHoYQ4y9PnhDbtH0EL3pvSYmDJ7sWS2JnizZ16-zF04PCG-ezuvs4cvtj5tv-d3-6_ebz3e5KRsx55pRXkvXWNO2ghtXC2e14bYC2ehWiJa11HUWGFSauqqUthOVKGVdVbrtZFNeZx827imGXwvgrEaftjAMeoKwoGJcliXlTLYpWm1REwNiBKdO0Y86ropRdVGjjmpToy5qFGMqqUlt754mLN0I9l_TXxcp8H4LOB2UPkSP6uE-EUTSVlZVfUF82hKQNnH2EBUaD5MB6yOYWdng__-H34zxrf8</recordid><startdate>20130125</startdate><enddate>20130125</enddate><creator>Vinnikova, Anna N.</creator><creator>Druzhinina, Tatyana N.</creator><creator>Danilov, Leonid L.</creator><creator>Utkina, Natalia S.</creator><creator>Torgov, Vladimir I.</creator><creator>Veselovsky, Vladimir V.</creator><creator>Wang, Shuo</creator><creator>Liu, Bin</creator><creator>Wang, Lei</creator><creator>Brockhausen, Inka</creator><general>Elsevier Ltd</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20130125</creationdate><title>Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5</title><author>Vinnikova, Anna N. ; Druzhinina, Tatyana N. ; Danilov, Leonid L. ; Utkina, Natalia S. ; Torgov, Vladimir I. ; Veselovsky, Vladimir V. ; Wang, Shuo ; Liu, Bin ; Wang, Lei ; Brockhausen, Inka</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-a10257f8dc9962cf56fdac2d4e78a9669190fbde1e4a0f437db64637544a9b783</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Biocatalysis</topic><topic>Chemical synthesis</topic><topic>E. coli O157</topic><topic>E. coli O5</topic><topic>Enterohemorrhagic Escherichia coli - enzymology</topic><topic>Enterohemorrhagic Escherichia coli - metabolism</topic><topic>Escherichia coli O157</topic><topic>Escherichia coli O157 - enzymology</topic><topic>Escherichia coli O157 - metabolism</topic><topic>fluorescence</topic><topic>Fluorescent assay</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Fluorescent Dyes - metabolism</topic><topic>genes</topic><topic>Glycosyltransferases</topic><topic>Glycosyltransferases - metabolism</topic><topic>Gram-negative bacteria</topic><topic>Molecular Structure</topic><topic>O Antigens - biosynthesis</topic><topic>O Antigens - chemistry</topic><topic>Stereoisomerism</topic><topic>sugars</topic><topic>WbdN</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Vinnikova, Anna N.</creatorcontrib><creatorcontrib>Druzhinina, Tatyana N.</creatorcontrib><creatorcontrib>Danilov, Leonid L.</creatorcontrib><creatorcontrib>Utkina, Natalia S.</creatorcontrib><creatorcontrib>Torgov, Vladimir I.</creatorcontrib><creatorcontrib>Veselovsky, Vladimir V.</creatorcontrib><creatorcontrib>Wang, Shuo</creatorcontrib><creatorcontrib>Liu, Bin</creatorcontrib><creatorcontrib>Wang, Lei</creatorcontrib><creatorcontrib>Brockhausen, Inka</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Carbohydrate research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vinnikova, Anna N.</au><au>Druzhinina, Tatyana N.</au><au>Danilov, Leonid L.</au><au>Utkina, Natalia S.</au><au>Torgov, Vladimir I.</au><au>Veselovsky, Vladimir V.</au><au>Wang, Shuo</au><au>Liu, Bin</au><au>Wang, Lei</au><au>Brockhausen, Inka</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5</atitle><jtitle>Carbohydrate research</jtitle><addtitle>Carbohydr Res</addtitle><date>2013-01-25</date><risdate>2013</risdate><volume>366</volume><issue>25</issue><spage>17</spage><epage>24</epage><pages>17-24</pages><issn>0008-6215</issn><eissn>1873-426X</eissn><abstract>[Display omitted]
► Fluorescent lipid diphosphate sugar. ► WbwC and WbdN substrate. ► Fluorescent assays.
The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-d-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars.</abstract><cop>Netherlands</cop><pub>Elsevier Ltd</pub><pmid>23261778</pmid><doi>10.1016/j.carres.2012.11.009</doi><tpages>8</tpages></addata></record> |
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| ispartof | Carbohydrate research, 2013-01, Vol.366 (25), p.17-24 |
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| subjects | Biocatalysis Chemical synthesis E. coli O157 E. coli O5 Enterohemorrhagic Escherichia coli - enzymology Enterohemorrhagic Escherichia coli - metabolism Escherichia coli O157 Escherichia coli O157 - enzymology Escherichia coli O157 - metabolism fluorescence Fluorescent assay Fluorescent Dyes - chemistry Fluorescent Dyes - metabolism genes Glycosyltransferases Glycosyltransferases - metabolism Gram-negative bacteria Molecular Structure O Antigens - biosynthesis O Antigens - chemistry Stereoisomerism sugars WbdN |
| title | Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5 |
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