Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase
Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISP ), a ferredoxin FER , and a ferredoxin reductase RED . RED and FER transfer electrons from NADH to an Fe-S active center of ISP wh...
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| Veröffentlicht in: | The Journal of biological chemistry 1996-04, Vol.271 (14), p.8152-8156 |
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| container_title | The Journal of biological chemistry |
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| creator | Hurtubise, Y Barriault, D Sylvestre, M |
| description | Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISP ), a ferredoxin FER , and a ferredoxin reductase RED . RED and FER transfer electrons from NADH to an Fe-S active center of ISP which activates molecular oxygen for insertion into the substrate. In this work B-356 ISP complex and its α and β subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISP construction carrying a single His tail on the N-terminal portion of the α subunit was active. Its major features were compared
to the untagged enzyme. In both cases, the native form is an α β heteromer, with each αβ unit containing a [2Fe-2S] Rieske center ( = 8,300 M cm ) and a mononuclear Fe . Although purified His-tagged α subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation
of this enzyme component and His-tagged β subunit to reconstitute active ISP was weak. However, when His-tagged α and β subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISP could be purified on Ni-nitrilotriacetic acid resin. |
| doi_str_mv | 10.1074/jbc.271.14.8152 |
| format | Article |
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to the untagged enzyme. In both cases, the native form is an α β heteromer, with each αβ unit containing a [2Fe-2S] Rieske center ( = 8,300 M cm ) and a mononuclear Fe . Although purified His-tagged α subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation
of this enzyme component and His-tagged β subunit to reconstitute active ISP was weak. However, when His-tagged α and β subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISP could be purified on Ni-nitrilotriacetic acid resin.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.271.14.8152</identifier><identifier>PMID: 8626504</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Base Sequence ; Comamonas testosteroni ; DNA Primers - chemistry ; Gram-Negative Aerobic Bacteria - chemistry ; Histidine - chemistry ; Kinetics ; Molecular Sequence Data ; Multienzyme Complexes - chemistry ; Oxygenases - chemistry ; Recombinant Proteins ; Spectrum Analysis ; Structure-Activity Relationship</subject><ispartof>The Journal of biological chemistry, 1996-04, Vol.271 (14), p.8152-8156</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c458t-304c74ce91a96b72e48683b7d7b4959ea46200c097b4c62ea3ea6ad1f88cff4a3</citedby><cites>FETCH-LOGICAL-c458t-304c74ce91a96b72e48683b7d7b4959ea46200c097b4c62ea3ea6ad1f88cff4a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8626504$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hurtubise, Y</creatorcontrib><creatorcontrib>Barriault, D</creatorcontrib><creatorcontrib>Sylvestre, M</creatorcontrib><title>Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISP ), a ferredoxin FER , and a ferredoxin reductase RED . RED and FER transfer electrons from NADH to an Fe-S active center of ISP which activates molecular oxygen for insertion into the substrate. In this work B-356 ISP complex and its α and β subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISP construction carrying a single His tail on the N-terminal portion of the α subunit was active. Its major features were compared
to the untagged enzyme. In both cases, the native form is an α β heteromer, with each αβ unit containing a [2Fe-2S] Rieske center ( = 8,300 M cm ) and a mononuclear Fe . Although purified His-tagged α subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation
of this enzyme component and His-tagged β subunit to reconstitute active ISP was weak. However, when His-tagged α and β subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISP could be purified on Ni-nitrilotriacetic acid resin.</description><subject>Base Sequence</subject><subject>Comamonas testosteroni</subject><subject>DNA Primers - chemistry</subject><subject>Gram-Negative Aerobic Bacteria - chemistry</subject><subject>Histidine - chemistry</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Multienzyme Complexes - chemistry</subject><subject>Oxygenases - chemistry</subject><subject>Recombinant Proteins</subject><subject>Spectrum Analysis</subject><subject>Structure-Activity Relationship</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtLAzEQx4MoWqtnT0JA8LY1yWbzOGp9VBAEUfAWsulsN9Ld1M1WrZ_eSKvgyVzCzP_BwA-hI0pGlEh-9lK6EZN0RPlI0YJtoQElKs_ygj5vowEhjGaaFWoP7cf4QtLjmu6iXSWYKAgfoM9xbTvreuj8p-19aHGo8Lnr_RvgB3ChKX1r2x5PfMx6O5vBFN9_rGbQ2gh4HJpFaCHJKZQG24S0xz3EPsRUGVqPL9ItAl_4RQ3tao4vffiJH6Cdys4jHG7-IXq6vnocT7K7-5vb8fld5nih-iwn3EnuQFOrRSkZcCVUXsqpLLkuNFguGCGO6DQ7wcDmYIWd0kopV1Xc5kN0uu5ddOF1mW4zjY8O5nPbQlhGIxVhSmv5r5FKwoTOVTKerY2uCzF2UJlF5xvbrQwl5huLSVhMwmIoN99YUuJ4U70sG5j--jcckn6y1ms_q999B6b0wdXQ_Gn5AgvVld0</recordid><startdate>19960405</startdate><enddate>19960405</enddate><creator>Hurtubise, Y</creator><creator>Barriault, D</creator><creator>Sylvestre, M</creator><general>American Society for Biochemistry and Molecular Biology</general><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>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>19960405</creationdate><title>Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase</title><author>Hurtubise, Y ; Barriault, D ; Sylvestre, M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c458t-304c74ce91a96b72e48683b7d7b4959ea46200c097b4c62ea3ea6ad1f88cff4a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Base Sequence</topic><topic>Comamonas testosteroni</topic><topic>DNA Primers - chemistry</topic><topic>Gram-Negative Aerobic Bacteria - chemistry</topic><topic>Histidine - chemistry</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Multienzyme Complexes - chemistry</topic><topic>Oxygenases - chemistry</topic><topic>Recombinant Proteins</topic><topic>Spectrum Analysis</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hurtubise, Y</creatorcontrib><creatorcontrib>Barriault, D</creatorcontrib><creatorcontrib>Sylvestre, M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hurtubise, Y</au><au>Barriault, D</au><au>Sylvestre, M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1996-04-05</date><risdate>1996</risdate><volume>271</volume><issue>14</issue><spage>8152</spage><epage>8156</epage><pages>8152-8156</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISP ), a ferredoxin FER , and a ferredoxin reductase RED . RED and FER transfer electrons from NADH to an Fe-S active center of ISP which activates molecular oxygen for insertion into the substrate. In this work B-356 ISP complex and its α and β subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISP construction carrying a single His tail on the N-terminal portion of the α subunit was active. Its major features were compared
to the untagged enzyme. In both cases, the native form is an α β heteromer, with each αβ unit containing a [2Fe-2S] Rieske center ( = 8,300 M cm ) and a mononuclear Fe . Although purified His-tagged α subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation
of this enzyme component and His-tagged β subunit to reconstitute active ISP was weak. However, when His-tagged α and β subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISP could be purified on Ni-nitrilotriacetic acid resin.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>8626504</pmid><doi>10.1074/jbc.271.14.8152</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | Base Sequence Comamonas testosteroni DNA Primers - chemistry Gram-Negative Aerobic Bacteria - chemistry Histidine - chemistry Kinetics Molecular Sequence Data Multienzyme Complexes - chemistry Oxygenases - chemistry Recombinant Proteins Spectrum Analysis Structure-Activity Relationship |
| title | Characterization of Active Recombinant His-tagged Oxygenase Component of Comamonas testosteroni B-356 Biphenyl Dioxygenase |
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