Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.
Identifieur interne : 000A04 ( PubMed/Curation ); précédent : 000A03; suivant : 000A05Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.
Auteurs : Daniel K. Owens [États-Unis] ; Cecilia A. McintoshSource :
- Phytochemistry [ 1873-3700 ]
English descriptors
- KwdEn :
- Amino Acid Sequence, Amino Acids (metabolism), Citrus paradisi (enzymology), Citrus paradisi (genetics), Citrus sinensis (enzymology), DNA, Complementary, Flavonoids (metabolism), Gene Expression, Genes, Plant, Glucosyltransferases (genetics), Glucosyltransferases (metabolism), Glycosylation, Metals (metabolism), Molecular Sequence Data, Recombinant Proteins (genetics), Recombinant Proteins (metabolism), Sequence Alignment, Substrate Specificity, Uridine Diphosphate.
- MESH :
- chemical , genetics : Glucosyltransferases, Recombinant Proteins.
- chemical , metabolism : Amino Acids, Flavonoids, Glucosyltransferases, Metals, Recombinant Proteins.
- enzymology : Citrus paradisi, Citrus sinensis.
- genetics : Citrus paradisi.
- Amino Acid Sequence, DNA, Complementary, Gene Expression, Genes, Plant, Glycosylation, Molecular Sequence Data, Sequence Alignment, Substrate Specificity, Uridine Diphosphate.
Abstract
Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.
DOI: 10.1016/j.phytochem.2009.07.027
PubMed: 19733370
Links toward previous steps (curation, corpus...)
- to stream PubMed, to step Corpus: Pour aller vers cette notice dans l'étape Curation :000A04
Links to Exploration step
pubmed:19733370Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.</title><author><name sortKey="Owens, Daniel K" sort="Owens, Daniel K" uniqKey="Owens D" first="Daniel K" last="Owens">Daniel K. Owens</name><affiliation wicri:level="1"><nlm:affiliation>School of Graduate Studies and Department of Biological Sciences, P.O. Box 70703, East Tennessee State University, Johnson City, TN 37614, United States.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>School of Graduate Studies and Department of Biological Sciences, P.O. Box 70703, East Tennessee State University, Johnson City, TN 37614</wicri:regionArea></affiliation></author><author><name sortKey="Mcintosh, Cecilia A" sort="Mcintosh, Cecilia A" uniqKey="Mcintosh C" first="Cecilia A" last="Mcintosh">Cecilia A. Mcintosh</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="????"><PubDate><MedlineDate>2009 Jul-Aug</MedlineDate></PubDate></date><idno type="RBID">pubmed:19733370</idno><idno type="pmid">19733370</idno><idno type="doi">10.1016/j.phytochem.2009.07.027</idno><idno type="wicri:Area/PubMed/Corpus">000A04</idno><idno type="wicri:Area/PubMed/Curation">000A04</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.</title><author><name sortKey="Owens, Daniel K" sort="Owens, Daniel K" uniqKey="Owens D" first="Daniel K" last="Owens">Daniel K. Owens</name><affiliation wicri:level="1"><nlm:affiliation>School of Graduate Studies and Department of Biological Sciences, P.O. Box 70703, East Tennessee State University, Johnson City, TN 37614, United States.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>School of Graduate Studies and Department of Biological Sciences, P.O. Box 70703, East Tennessee State University, Johnson City, TN 37614</wicri:regionArea></affiliation></author><author><name sortKey="Mcintosh, Cecilia A" sort="Mcintosh, Cecilia A" uniqKey="Mcintosh C" first="Cecilia A" last="Mcintosh">Cecilia A. Mcintosh</name></author></analytic><series><title level="j">Phytochemistry</title><idno type="eISSN">1873-3700</idno></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term><term>Amino Acids (metabolism)</term><term>Citrus paradisi (enzymology)</term><term>Citrus paradisi (genetics)</term><term>Citrus sinensis (enzymology)</term><term>DNA, Complementary</term><term>Flavonoids (metabolism)</term><term>Gene Expression</term><term>Genes, Plant</term><term>Glucosyltransferases (genetics)</term><term>Glucosyltransferases (metabolism)</term><term>Glycosylation</term><term>Metals (metabolism)</term><term>Molecular Sequence Data</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (metabolism)</term><term>Sequence Alignment</term><term>Substrate Specificity</term><term>Uridine Diphosphate</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Glucosyltransferases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Amino Acids</term><term>Flavonoids</term><term>Glucosyltransferases</term><term>Metals</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Citrus paradisi</term><term>Citrus sinensis</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Citrus paradisi</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>DNA, Complementary</term><term>Gene Expression</term><term>Genes, Plant</term><term>Glycosylation</term><term>Molecular Sequence Data</term><term>Sequence Alignment</term><term>Substrate Specificity</term><term>Uridine Diphosphate</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">19733370</PMID><DateCreated><Year>2009</Year><Month>9</Month><Day>29</Day></DateCreated><DateCompleted><Year>2010</Year><Month>01</Month><Day>13</Day></DateCompleted><DateRevised><Year>2009</Year><Month>9</Month><Day>29</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1873-3700</ISSN><JournalIssue CitedMedium="Internet"><Volume>70</Volume><Issue>11-12</Issue><PubDate><MedlineDate>2009 Jul-Aug</MedlineDate></PubDate></JournalIssue><Title>Phytochemistry</Title><ISOAbbreviation>Phytochemistry</ISOAbbreviation></Journal><ArticleTitle>Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.</ArticleTitle><Pagination><MedlinePgn>1382-91</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.phytochem.2009.07.027</ELocationID><Abstract><AbstractText>Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Owens</LastName><ForeName>Daniel K</ForeName><Initials>DK</Initials><AffiliationInfo><Affiliation>School of Graduate Studies and Department of Biological Sciences, P.O. Box 70703, East Tennessee State University, Johnson City, TN 37614, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>McIntosh</LastName><ForeName>Cecilia A</ForeName><Initials>CA</Initials></Author></AuthorList><Language>ENG</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2009</Year><Month>Sep</Month><Day>03</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Phytochemistry</MedlineTA><NlmUniqueID>0151434</NlmUniqueID><ISSNLinking>0031-9422</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000596">Amino Acids</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D018076">DNA, Complementary</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005419">Flavonoids</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D008670">Metals</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>58-98-0</RegistryNumber><NameOfSubstance UI="D014530">Uridine Diphosphate</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.4.1.-</RegistryNumber><NameOfSubstance UI="D005964">Glucosyltransferases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.4.1.91</RegistryNumber><NameOfSubstance UI="C023249">flavonol 3-O-glucosyltransferase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000595" MajorTopicYN="N">Amino Acid Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000596" MajorTopicYN="N">Amino Acids</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D032083" MajorTopicYN="N">Citrus paradisi</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D032084" MajorTopicYN="N">Citrus sinensis</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="N">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018076" MajorTopicYN="N">DNA, Complementary</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005419" MajorTopicYN="N">Flavonoids</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="Y">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017343" MajorTopicYN="Y">Genes, Plant</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005964" MajorTopicYN="N">Glucosyltransferases</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006031" MajorTopicYN="N">Glycosylation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008670" MajorTopicYN="N">Metals</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016415" MajorTopicYN="N">Sequence Alignment</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013379" MajorTopicYN="N">Substrate Specificity</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014530" MajorTopicYN="N">Uridine Diphosphate</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2009</Year><Month>2</Month><Day>24</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2009</Year><Month>5</Month><Day>23</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2009</Year><Month>7</Month><Day>22</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2009</Year><Month>9</Month><Day>8</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2009</Year><Month>9</Month><Day>8</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2010</Year><Month>1</Month><Day>14</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">19733370</ArticleId><ArticleId IdType="pii">S0031-9422(09)00303-3</ArticleId><ArticleId IdType="doi">10.1016/j.phytochem.2009.07.027</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Bois/explor/OrangerV1/Data/PubMed/Curation
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000A04 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/PubMed/Curation/biblio.hfd -nk 000A04 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Bois
|area= OrangerV1
|flux= PubMed
|étape= Curation
|type= RBID
|clé= pubmed:19733370
|texte= Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/PubMed/Curation/RBID.i -Sk "pubmed:19733370" \
| HfdSelect -Kh $EXPLOR_AREA/Data/PubMed/Curation/biblio.hfd \
| NlmPubMed2Wicri -a OrangerV1
| This area was generated with Dilib version V0.6.25. |  |