Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.
Identifieur interne : 000435 ( Main/Exploration ); précédent : 000434; suivant : 000436Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.
Auteurs : Hirofumi Ichinose [Japon] ; Hiroyuki WariishiSource :
- Archives of biochemistry and biophysics [ 1096-0384 ] ; 2012.
Descripteurs français
- KwdFr :
- Activation enzymatique (MeSH), Analyse spectrale (MeSH), Biocatalyse (MeSH), Cytochrome P-450 enzyme system (composition chimique), Cytochrome P-450 enzyme system (génétique), Cytochrome P-450 enzyme system (isolement et purification), Cytochrome P-450 enzyme system (métabolisme), Cytochrome-B(5) reductase (métabolisme), Cytochromes b5 (métabolisme), Données de séquences moléculaires (MeSH), Escherichia coli (génétique), Expression des gènes (MeSH), NAD (métabolisme), Oxydoréduction (MeSH), Phanerochaete (enzymologie), Phanerochaete (génétique), Séquence d'acides aminés (MeSH).
- MESH :
- composition chimique : Cytochrome P-450 enzyme system.
- enzymologie : Phanerochaete.
- génétique : Cytochrome P-450 enzyme system, Escherichia coli, Phanerochaete.
- isolement et purification : Cytochrome P-450 enzyme system.
- métabolisme : Cytochrome P-450 enzyme system, Cytochrome-B(5) reductase, Cytochromes b5, NAD.
- Activation enzymatique, Analyse spectrale, Biocatalyse, Données de séquences moléculaires, Expression des gènes, Oxydoréduction, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence (MeSH), Biocatalysis (MeSH), Cytochrome P-450 Enzyme System (chemistry), Cytochrome P-450 Enzyme System (genetics), Cytochrome P-450 Enzyme System (isolation & purification), Cytochrome P-450 Enzyme System (metabolism), Cytochrome-B(5) Reductase (metabolism), Cytochromes b5 (metabolism), Enzyme Activation (MeSH), Escherichia coli (genetics), Gene Expression (MeSH), Molecular Sequence Data (MeSH), NAD (metabolism), Oxidation-Reduction (MeSH), Phanerochaete (enzymology), Phanerochaete (genetics), Spectrum Analysis (MeSH).
- MESH :
- chemical , chemistry : Cytochrome P-450 Enzyme System.
- chemical , genetics : Cytochrome P-450 Enzyme System.
- chemical , isolation & purification : Cytochrome P-450 Enzyme System.
- chemical , metabolism : Cytochrome P-450 Enzyme System, Cytochrome-B(5) Reductase, Cytochromes b5, NAD.
- enzymology : Phanerochaete.
- genetics : Escherichia coli, Phanerochaete.
- Amino Acid Sequence, Biocatalysis, Enzyme Activation, Gene Expression, Molecular Sequence Data, Oxidation-Reduction, Spectrum Analysis.
Abstract
A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.
DOI: 10.1016/j.abb.2011.12.010
PubMed: 22206618
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.</title><author><name sortKey="Ichinose, Hirofumi" sort="Ichinose, Hirofumi" uniqKey="Ichinose H" first="Hirofumi" last="Ichinose">Hirofumi Ichinose</name><affiliation wicri:level="4"><nlm:affiliation>Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan. ichinose@agr.kyushu-u.ac.jp</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Kyushu University, Hakozaki, Higashi-ku, Fukuoka</wicri:regionArea><orgName type="university">Université de Kyūshū</orgName><placeName><settlement type="city">Fukuoka</settlement><region type="province">Kyūshū</region><region type="prefecture">Préfecture de Fukuoka</region></placeName></affiliation></author><author><name sortKey="Wariishi, Hiroyuki" sort="Wariishi, Hiroyuki" uniqKey="Wariishi H" first="Hiroyuki" last="Wariishi">Hiroyuki Wariishi</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2012">2012</date><idno type="RBID">pubmed:22206618</idno><idno type="pmid">22206618</idno><idno type="doi">10.1016/j.abb.2011.12.010</idno><idno type="wicri:Area/Main/Corpus">000461</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000461</idno><idno type="wicri:Area/Main/Curation">000461</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">000461</idno><idno type="wicri:Area/Main/Exploration">000461</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.</title><author><name sortKey="Ichinose, Hirofumi" sort="Ichinose, Hirofumi" uniqKey="Ichinose H" first="Hirofumi" last="Ichinose">Hirofumi Ichinose</name><affiliation wicri:level="4"><nlm:affiliation>Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan. ichinose@agr.kyushu-u.ac.jp</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Kyushu University, Hakozaki, Higashi-ku, Fukuoka</wicri:regionArea><orgName type="university">Université de Kyūshū</orgName><placeName><settlement type="city">Fukuoka</settlement><region type="province">Kyūshū</region><region type="prefecture">Préfecture de Fukuoka</region></placeName></affiliation></author><author><name sortKey="Wariishi, Hiroyuki" sort="Wariishi, Hiroyuki" uniqKey="Wariishi H" first="Hiroyuki" last="Wariishi">Hiroyuki Wariishi</name></author></analytic><series><title level="j">Archives of biochemistry and biophysics</title><idno type="eISSN">1096-0384</idno><imprint><date when="2012" type="published">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence (MeSH)</term><term>Biocatalysis (MeSH)</term><term>Cytochrome P-450 Enzyme System (chemistry)</term><term>Cytochrome P-450 Enzyme System (genetics)</term><term>Cytochrome P-450 Enzyme System (isolation & purification)</term><term>Cytochrome P-450 Enzyme System (metabolism)</term><term>Cytochrome-B(5) Reductase (metabolism)</term><term>Cytochromes b5 (metabolism)</term><term>Enzyme Activation (MeSH)</term><term>Escherichia coli (genetics)</term><term>Gene Expression (MeSH)</term><term>Molecular Sequence Data (MeSH)</term><term>NAD (metabolism)</term><term>Oxidation-Reduction (MeSH)</term><term>Phanerochaete (enzymology)</term><term>Phanerochaete (genetics)</term><term>Spectrum Analysis (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Activation enzymatique (MeSH)</term><term>Analyse spectrale (MeSH)</term><term>Biocatalyse (MeSH)</term><term>Cytochrome P-450 enzyme system (composition chimique)</term><term>Cytochrome P-450 enzyme system (génétique)</term><term>Cytochrome P-450 enzyme system (isolement et purification)</term><term>Cytochrome P-450 enzyme system (métabolisme)</term><term>Cytochrome-B(5) reductase (métabolisme)</term><term>Cytochromes b5 (métabolisme)</term><term>Données de séquences moléculaires (MeSH)</term><term>Escherichia coli (génétique)</term><term>Expression des gènes (MeSH)</term><term>NAD (métabolisme)</term><term>Oxydoréduction (MeSH)</term><term>Phanerochaete (enzymologie)</term><term>Phanerochaete (génétique)</term><term>Séquence d'acides aminés (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Cytochrome P-450 Enzyme System</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cytochrome P-450 Enzyme System</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Cytochrome P-450 Enzyme System</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cytochrome P-450 Enzyme System</term><term>Cytochrome-B(5) Reductase</term><term>Cytochromes b5</term><term>NAD</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Cytochrome P-450 enzyme system</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cytochrome P-450 enzyme system</term><term>Escherichia coli</term><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Cytochrome P-450 enzyme system</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Cytochrome P-450 enzyme system</term><term>Cytochrome-B(5) reductase</term><term>Cytochromes b5</term><term>NAD</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Biocatalysis</term><term>Enzyme Activation</term><term>Gene Expression</term><term>Molecular Sequence Data</term><term>Oxidation-Reduction</term><term>Spectrum Analysis</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Activation enzymatique</term><term>Analyse spectrale</term><term>Biocatalyse</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Oxydoréduction</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">22206618</PMID><DateCompleted><Year>2012</Year><Month>03</Month><Day>05</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1096-0384</ISSN><JournalIssue CitedMedium="Internet"><Volume>518</Volume><Issue>1</Issue><PubDate><Year>2012</Year><Month>Feb</Month><Day>01</Day></PubDate></JournalIssue><Title>Archives of biochemistry and biophysics</Title><ISOAbbreviation>Arch Biochem Biophys</ISOAbbreviation></Journal><ArticleTitle>Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.</ArticleTitle><Pagination><MedlinePgn>8-15</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.abb.2011.12.010</ELocationID><Abstract><AbstractText>A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.</AbstractText><CopyrightInformation>Copyright © 2011 Elsevier Inc. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Ichinose</LastName><ForeName>Hirofumi</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan. ichinose@agr.kyushu-u.ac.jp</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wariishi</LastName><ForeName>Hiroyuki</ForeName><Initials>H</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2011</Year><Month>12</Month><Day>20</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Arch Biochem Biophys</MedlineTA><NlmUniqueID>0372430</NlmUniqueID><ISSNLinking>0003-9861</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0U46U6E8UK</RegistryNumber><NameOfSubstance UI="D009243">NAD</NameOfSubstance></Chemical><Chemical><RegistryNumber>9035-39-6</RegistryNumber><NameOfSubstance UI="D015786">Cytochromes b5</NameOfSubstance></Chemical><Chemical><RegistryNumber>9035-51-2</RegistryNumber><NameOfSubstance UI="D003577">Cytochrome P-450 Enzyme System</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.6.2.2</RegistryNumber><NameOfSubstance UI="D042966">Cytochrome-B(5) Reductase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000595" MajorTopicYN="N">Amino Acid Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D055162" MajorTopicYN="N">Biocatalysis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003577" MajorTopicYN="N">Cytochrome P-450 Enzyme System</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D042966" MajorTopicYN="N">Cytochrome-B(5) Reductase</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015786" MajorTopicYN="N">Cytochromes b5</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004789" MajorTopicYN="N">Enzyme Activation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015870" MajorTopicYN="N">Gene Expression</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D009243" MajorTopicYN="N">NAD</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010084" MajorTopicYN="N">Oxidation-Reduction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D020075" MajorTopicYN="N">Phanerochaete</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013057" MajorTopicYN="N">Spectrum Analysis</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2011</Year><Month>11</Month><Day>07</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2011</Year><Month>12</Month><Day>13</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2011</Year><Month>12</Month><Day>13</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2011</Year><Month>12</Month><Day>31</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2011</Year><Month>12</Month><Day>31</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2012</Year><Month>3</Month><Day>6</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">22206618</ArticleId><ArticleId IdType="pii">S0003-9861(11)00413-9</ArticleId><ArticleId IdType="doi">10.1016/j.abb.2011.12.010</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Japon</li></country><region><li>Kyūshū</li><li>Préfecture de Fukuoka</li></region><settlement><li>Fukuoka</li></settlement><orgName><li>Université de Kyūshū</li></orgName></list><tree><noCountry><name sortKey="Wariishi, Hiroyuki" sort="Wariishi, Hiroyuki" uniqKey="Wariishi H" first="Hiroyuki" last="Wariishi">Hiroyuki Wariishi</name></noCountry><country name="Japon"><region name="Kyūshū"><name sortKey="Ichinose, Hirofumi" sort="Ichinose, Hirofumi" uniqKey="Ichinose H" first="Hirofumi" last="Ichinose">Hirofumi Ichinose</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Bois/explor/PhanerochaeteV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000435 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 000435 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Bois
|area= PhanerochaeteV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= pubmed:22206618
|texte= Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i -Sk "pubmed:22206618" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a PhanerochaeteV1
| This area was generated with Dilib version V0.6.37. |  |