Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit
•MS provides valuable information about in vitro characterization of glycosyltransferases.•MS is highly valuable for precise structure elucidation of lipooligosaccharides.•The wbuP gene of E. coli O114 encodes a new β-1,3-Gal-transferase.•Nonionic surfactants exert a strong inhibitory effect on the...
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| Veröffentlicht in: | Carbohydrate research 2013-11, Vol.381, p.43-50 |
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| creator | Zhou, Dawei Utkina, Natalia Li, Diange Dong, Chenying Druzhinina, Tatyana Veselovsky, Vladimir Liu, Bin |
| description | •MS provides valuable information about in vitro characterization of glycosyltransferases.•MS is highly valuable for precise structure elucidation of lipooligosaccharides.•The wbuP gene of E. coli O114 encodes a new β-1,3-Gal-transferase.•Nonionic surfactants exert a strong inhibitory effect on the activity of WbuP.
In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn2+ ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit. |
| doi_str_mv | 10.1016/j.carres.2013.08.021 |
| format | Article |
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In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn2+ ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.</description><identifier>ISSN: 0008-6215</identifier><identifier>EISSN: 1873-426X</identifier><identifier>DOI: 10.1016/j.carres.2013.08.021</identifier><identifier>PMID: 24056013</identifier><language>eng</language><publisher>Netherlands: Elsevier Ltd</publisher><subject>Biocatalysis ; Carbohydrate Conformation ; desorption ; detergents ; digestion ; dissociation ; Enzyme Activation ; enzyme activity ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli O-antigen ; Functional characterization ; Galactosyltransferases - chemistry ; Galactosyltransferases - genetics ; Galactosyltransferases - metabolism ; genes ; Glycosyltransferase ; ionization ; ions ; Kinetics ; manganese ; Mass spectrometry ; matrix-assisted laser desorption-ionization mass spectrometry ; Molecular Sequence Data ; O Antigens - chemistry ; O Antigens - metabolism ; sugars</subject><ispartof>Carbohydrate research, 2013-11, Vol.381, p.43-50</ispartof><rights>2013 Elsevier Ltd</rights><rights>Copyright © 2013 Elsevier Ltd. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-2466d45c43b74e112b65b14d618d660e0489abe86c771a94c9e92e6efc4a80853</citedby><cites>FETCH-LOGICAL-c386t-2466d45c43b74e112b65b14d618d660e0489abe86c771a94c9e92e6efc4a80853</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.carres.2013.08.021$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24056013$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhou, Dawei</creatorcontrib><creatorcontrib>Utkina, Natalia</creatorcontrib><creatorcontrib>Li, Diange</creatorcontrib><creatorcontrib>Dong, Chenying</creatorcontrib><creatorcontrib>Druzhinina, Tatyana</creatorcontrib><creatorcontrib>Veselovsky, Vladimir</creatorcontrib><creatorcontrib>Liu, Bin</creatorcontrib><title>Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit</title><title>Carbohydrate research</title><addtitle>Carbohydr Res</addtitle><description>•MS provides valuable information about in vitro characterization of glycosyltransferases.•MS is highly valuable for precise structure elucidation of lipooligosaccharides.•The wbuP gene of E. coli O114 encodes a new β-1,3-Gal-transferase.•Nonionic surfactants exert a strong inhibitory effect on the activity of WbuP.
In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn2+ ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.</description><subject>Biocatalysis</subject><subject>Carbohydrate Conformation</subject><subject>desorption</subject><subject>detergents</subject><subject>digestion</subject><subject>dissociation</subject><subject>Enzyme Activation</subject><subject>enzyme activity</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli O-antigen</subject><subject>Functional characterization</subject><subject>Galactosyltransferases - chemistry</subject><subject>Galactosyltransferases - genetics</subject><subject>Galactosyltransferases - metabolism</subject><subject>genes</subject><subject>Glycosyltransferase</subject><subject>ionization</subject><subject>ions</subject><subject>Kinetics</subject><subject>manganese</subject><subject>Mass spectrometry</subject><subject>matrix-assisted laser desorption-ionization mass spectrometry</subject><subject>Molecular Sequence Data</subject><subject>O Antigens - chemistry</subject><subject>O Antigens - metabolism</subject><subject>sugars</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>eNp9kc1u1DAURi0EokPhDRB4yaIJduI4zgYJqvIjVRokqGBn3Tg3Mx5l7KntUE3fhZfgQXgmXKWwZGVd-XzftXwIec5ZyRmXr3elgRAwlhXjdclUySr-gKy4autCVPL7Q7JijKlCVrw5IU9i3OWRyVY-JieVYI3MsRX5-c56s8W9NTBRs4UAJmGwt5Csd9SPFKjDG_r7V8HP6mIDU7738TilAC6OGCAi_dbPn-kY_J5exNwVrNlaoMZPlq45FzRtIVEDCabjLcY8Io1ovBtoTHig1tF1AS7ZDToa8IB5t9sUs7PpKXk0whTx2f15Sq7eX3w9_1hcrj98On97WZhayVRUQspBNEbUfSuQ86qXTc_FILkapGTIhOqgRyVN23LohOmwq1DiaAQoppr6lLxaeg_BX88Yk97baHCawKGfo-ZCiLqrleAZFQtqgo8x4KgPwe4hHDVn-k6M3ulFjL4To5nSWUyOvbjfMPd7HP6F_prIwMsFGMFr2AQb9dWX3CCztFrItsvEm4XA_BM_LAYdjUVncLABTdKDt_9_wx_SI6wA</recordid><startdate>20131115</startdate><enddate>20131115</enddate><creator>Zhou, Dawei</creator><creator>Utkina, Natalia</creator><creator>Li, Diange</creator><creator>Dong, Chenying</creator><creator>Druzhinina, Tatyana</creator><creator>Veselovsky, Vladimir</creator><creator>Liu, Bin</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>20131115</creationdate><title>Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit</title><author>Zhou, Dawei ; Utkina, Natalia ; Li, Diange ; Dong, Chenying ; Druzhinina, Tatyana ; Veselovsky, Vladimir ; Liu, Bin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-2466d45c43b74e112b65b14d618d660e0489abe86c771a94c9e92e6efc4a80853</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Biocatalysis</topic><topic>Carbohydrate Conformation</topic><topic>desorption</topic><topic>detergents</topic><topic>digestion</topic><topic>dissociation</topic><topic>Enzyme Activation</topic><topic>enzyme activity</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli O-antigen</topic><topic>Functional characterization</topic><topic>Galactosyltransferases - chemistry</topic><topic>Galactosyltransferases - genetics</topic><topic>Galactosyltransferases - metabolism</topic><topic>genes</topic><topic>Glycosyltransferase</topic><topic>ionization</topic><topic>ions</topic><topic>Kinetics</topic><topic>manganese</topic><topic>Mass spectrometry</topic><topic>matrix-assisted laser desorption-ionization mass spectrometry</topic><topic>Molecular Sequence Data</topic><topic>O Antigens - chemistry</topic><topic>O Antigens - metabolism</topic><topic>sugars</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zhou, Dawei</creatorcontrib><creatorcontrib>Utkina, Natalia</creatorcontrib><creatorcontrib>Li, Diange</creatorcontrib><creatorcontrib>Dong, Chenying</creatorcontrib><creatorcontrib>Druzhinina, Tatyana</creatorcontrib><creatorcontrib>Veselovsky, Vladimir</creatorcontrib><creatorcontrib>Liu, Bin</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>Zhou, Dawei</au><au>Utkina, Natalia</au><au>Li, Diange</au><au>Dong, Chenying</au><au>Druzhinina, Tatyana</au><au>Veselovsky, Vladimir</au><au>Liu, Bin</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit</atitle><jtitle>Carbohydrate research</jtitle><addtitle>Carbohydr Res</addtitle><date>2013-11-15</date><risdate>2013</risdate><volume>381</volume><spage>43</spage><epage>50</epage><pages>43-50</pages><issn>0008-6215</issn><eissn>1873-426X</eissn><abstract>•MS provides valuable information about in vitro characterization of glycosyltransferases.•MS is highly valuable for precise structure elucidation of lipooligosaccharides.•The wbuP gene of E. coli O114 encodes a new β-1,3-Gal-transferase.•Nonionic surfactants exert a strong inhibitory effect on the activity of WbuP.
In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn2+ ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.</abstract><cop>Netherlands</cop><pub>Elsevier Ltd</pub><pmid>24056013</pmid><doi>10.1016/j.carres.2013.08.021</doi><tpages>8</tpages></addata></record> |
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| ispartof | Carbohydrate research, 2013-11, Vol.381, p.43-50 |
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| subjects | Biocatalysis Carbohydrate Conformation desorption detergents digestion dissociation Enzyme Activation enzyme activity Escherichia coli Escherichia coli - enzymology Escherichia coli O-antigen Functional characterization Galactosyltransferases - chemistry Galactosyltransferases - genetics Galactosyltransferases - metabolism genes Glycosyltransferase ionization ions Kinetics manganese Mass spectrometry matrix-assisted laser desorption-ionization mass spectrometry Molecular Sequence Data O Antigens - chemistry O Antigens - metabolism sugars |
| title | Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit |
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