Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta
Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms...
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Veröffentlicht in: | Biochemical journal 2003-08, Vol.373 (Pt 3), p.987-992 |
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description | Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations. |
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The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.</description><identifier>ISSN: 0264-6021</identifier><identifier>EISSN: 1470-8728</identifier><identifier>DOI: 10.1042/bj20021453</identifier><identifier>PMID: 12741958</identifier><language>eng</language><publisher>England</publisher><subject>Arabidopsis - enzymology ; Arabidopsis - metabolism ; Base Sequence ; Caffeic Acids - metabolism ; Chromatography, High Pressure Liquid ; DNA Primers ; Glucosyltransferases - metabolism ; Glycosylation ; Molecular Sequence Data ; Nuclear Magnetic Resonance, Biomolecular ; Plants, Genetically Modified ; Substrate Specificity</subject><ispartof>Biochemical journal, 2003-08, Vol.373 (Pt 3), p.987-992</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c481t-5a1c8c2aed067babb63b3144c32939dd1f024a9ac6a73cac5a133a1661f089063</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223554/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223554/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12741958$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lim, Eng-Kiat</creatorcontrib><creatorcontrib>Higgins, Gillian S</creatorcontrib><creatorcontrib>Li, Yi</creatorcontrib><creatorcontrib>Bowles, Dianna J</creatorcontrib><title>Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta</title><title>Biochemical journal</title><addtitle>Biochem J</addtitle><description>Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.</description><subject>Arabidopsis - enzymology</subject><subject>Arabidopsis - metabolism</subject><subject>Base Sequence</subject><subject>Caffeic Acids - metabolism</subject><subject>Chromatography, High Pressure Liquid</subject><subject>DNA Primers</subject><subject>Glucosyltransferases - metabolism</subject><subject>Glycosylation</subject><subject>Molecular Sequence Data</subject><subject>Nuclear Magnetic Resonance, Biomolecular</subject><subject>Plants, Genetically Modified</subject><subject>Substrate Specificity</subject><issn>0264-6021</issn><issn>1470-8728</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkV1LwzAYhYMobn7c-AMkV14I1Xw1bb0QZH7CQBF3Hd6m6cxom9m0g_57MzadXoSQ9zyc94SD0BklV5QIdp0vGCGMipjvoTEVCYnShKX7aEyYFJEM0ggdeb8ghAoiyCEaUZYImsXpGDXvZm6dN5XRnV3ZbsCuxPOq184PFXTWNeuBhrI0VmPQtsD5gAHP7t-iDWZufvCuhcaXpgVvsPW4Btt04ZgC2wYvKwivE3RQQuXN6fY-RrPHh4_JczR9fXqZ3E0jLVLaRTFQnWoGpiAyySHPJc85FUJzlvGsKGhJmIAMtISEa9CB5xyolEFIMyL5Mbrd-C77vDaFNk0IV6lla2toB-XAqv9KYz_V3K0UZYzHsQgGF1uD1n31xneqtl6bKvzCuN6rJFBJwmkALzegbp33rSl_l1Ci1vWoXT0BPv8ba4du--DfN_ONrg</recordid><startdate>20030801</startdate><enddate>20030801</enddate><creator>Lim, Eng-Kiat</creator><creator>Higgins, Gillian S</creator><creator>Li, Yi</creator><creator>Bowles, Dianna J</creator><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><scope>5PM</scope></search><sort><creationdate>20030801</creationdate><title>Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta</title><author>Lim, Eng-Kiat ; Higgins, Gillian S ; Li, Yi ; Bowles, Dianna J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c481t-5a1c8c2aed067babb63b3144c32939dd1f024a9ac6a73cac5a133a1661f089063</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Arabidopsis - enzymology</topic><topic>Arabidopsis - metabolism</topic><topic>Base Sequence</topic><topic>Caffeic Acids - metabolism</topic><topic>Chromatography, High Pressure Liquid</topic><topic>DNA Primers</topic><topic>Glucosyltransferases - metabolism</topic><topic>Glycosylation</topic><topic>Molecular Sequence Data</topic><topic>Nuclear Magnetic Resonance, Biomolecular</topic><topic>Plants, Genetically Modified</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lim, Eng-Kiat</creatorcontrib><creatorcontrib>Higgins, Gillian S</creatorcontrib><creatorcontrib>Li, Yi</creatorcontrib><creatorcontrib>Bowles, Dianna J</creatorcontrib><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><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biochemical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lim, Eng-Kiat</au><au>Higgins, Gillian S</au><au>Li, Yi</au><au>Bowles, Dianna J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta</atitle><jtitle>Biochemical journal</jtitle><addtitle>Biochem J</addtitle><date>2003-08-01</date><risdate>2003</risdate><volume>373</volume><issue>Pt 3</issue><spage>987</spage><epage>992</epage><pages>987-992</pages><issn>0264-6021</issn><eissn>1470-8728</eissn><abstract>Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.</abstract><cop>England</cop><pmid>12741958</pmid><doi>10.1042/bj20021453</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Arabidopsis - enzymology Arabidopsis - metabolism Base Sequence Caffeic Acids - metabolism Chromatography, High Pressure Liquid DNA Primers Glucosyltransferases - metabolism Glycosylation Molecular Sequence Data Nuclear Magnetic Resonance, Biomolecular Plants, Genetically Modified Substrate Specificity |
title | Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta |
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