Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair
Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH−). After purification in vitro, the cofactor FADH− in photolyase is oxidized into the...
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| Veröffentlicht in: | Biochemistry (Easton) 2008-08, Vol.47 (33), p.8736-8743 |
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| container_title | Biochemistry (Easton) |
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| creator | Xu, Lei Mu, Wanmeng Ding, Yanwei Luo, Zhaofeng Han, Qingkai Bi, Fuyong Wang, Yuzhen Song, Qinhua |
| description | Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH−). After purification in vitro, the cofactor FADH− in photolyase is oxidized into the neutral radical form FADH• under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FADox) but not FADH• after routine purification procedures, but evidence shows that the mutant protein contains FADH− in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis. |
| doi_str_mv | 10.1021/bi800391j |
| format | Article |
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The catalytic activity of the enzyme requires fully reduced FAD (FADH−). After purification in vitro, the cofactor FADH− in photolyase is oxidized into the neutral radical form FADH• under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FADox) but not FADH• after routine purification procedures, but evidence shows that the mutant protein contains FADH− in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi800391j</identifier><identifier>PMID: 18652481</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Asparagine - chemistry ; Binding Sites ; Deoxyribodipyrimidine Photo-Lyase - chemistry ; Deoxyribodipyrimidine Photo-Lyase - metabolism ; DNA Damage ; DNA Repair - physiology ; DNA, Bacterial ; Escherichia coli - enzymology ; Flavins - chemistry ; Mutation ; Protein Conformation</subject><ispartof>Biochemistry (Easton), 2008-08, Vol.47 (33), p.8736-8743</ispartof><rights>Copyright © 2008 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a417t-c00ed48826ce3d6afac7c45eac44e730090f41c3175451a1bc558c5d447426473</citedby><cites>FETCH-LOGICAL-a417t-c00ed48826ce3d6afac7c45eac44e730090f41c3175451a1bc558c5d447426473</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi800391j$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi800391j$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>315,781,785,2766,27081,27929,27930,56743,56793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18652481$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Xu, Lei</creatorcontrib><creatorcontrib>Mu, Wanmeng</creatorcontrib><creatorcontrib>Ding, Yanwei</creatorcontrib><creatorcontrib>Luo, Zhaofeng</creatorcontrib><creatorcontrib>Han, Qingkai</creatorcontrib><creatorcontrib>Bi, Fuyong</creatorcontrib><creatorcontrib>Wang, Yuzhen</creatorcontrib><creatorcontrib>Song, Qinhua</creatorcontrib><title>Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH−). After purification in vitro, the cofactor FADH− in photolyase is oxidized into the neutral radical form FADH• under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FADox) but not FADH• after routine purification procedures, but evidence shows that the mutant protein contains FADH− in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.</description><subject>Asparagine - chemistry</subject><subject>Binding Sites</subject><subject>Deoxyribodipyrimidine Photo-Lyase - chemistry</subject><subject>Deoxyribodipyrimidine Photo-Lyase - metabolism</subject><subject>DNA Damage</subject><subject>DNA Repair - physiology</subject><subject>DNA, Bacterial</subject><subject>Escherichia coli - enzymology</subject><subject>Flavins - chemistry</subject><subject>Mutation</subject><subject>Protein Conformation</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkM1uEzEURi0EomlhwQsgb0DqYqg9458ZdlHahqIoVElZWx7PHcZhMg62p2p5B94Zh0TthtXV1T33-6SD0DtKPlGS04valoQUFd28QBPKc5KxquIv0YQQIrK8EuQEnYawSSsjkr1GJ7QUPGclnaA_UxPtPeC1jYBdi6-C6cBb01mNjestvlxO8W3nousfdYDPeBqGQpb4JuCZH43VPa5d7HDrPF5HXdve_rbDDxw7wEsYo0_Ada_v7YBXurEmrTPXahMTr4fm39--YgU7bf0b9KrVfYC3x3mGvl9f3c2-ZItv85vZdJFpRmXMDCHQsLLMhYGiETrlScM4aMMYyIKQirSMmoJKzjjVtDacl4Y3jEmWCyaLM_TxkLvz7tcIIaqtDQb6Xg_gxqBExajISZHA8wNovAvBQ6t23m61f1SUqL179eQ-se-PoWO9heaZPMpOQHYAbIjw8HTX_qcSspBc3d2u1bycr77y5ULtyz8ceG2C2rjRD8nJf4r_AiItmKU</recordid><startdate>20080819</startdate><enddate>20080819</enddate><creator>Xu, Lei</creator><creator>Mu, Wanmeng</creator><creator>Ding, Yanwei</creator><creator>Luo, Zhaofeng</creator><creator>Han, Qingkai</creator><creator>Bi, Fuyong</creator><creator>Wang, Yuzhen</creator><creator>Song, Qinhua</creator><general>American Chemical Society</general><scope>BSCLL</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>20080819</creationdate><title>Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair</title><author>Xu, Lei ; Mu, Wanmeng ; Ding, Yanwei ; Luo, Zhaofeng ; Han, Qingkai ; Bi, Fuyong ; Wang, Yuzhen ; Song, Qinhua</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a417t-c00ed48826ce3d6afac7c45eac44e730090f41c3175451a1bc558c5d447426473</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Asparagine - chemistry</topic><topic>Binding Sites</topic><topic>Deoxyribodipyrimidine Photo-Lyase - chemistry</topic><topic>Deoxyribodipyrimidine Photo-Lyase - metabolism</topic><topic>DNA Damage</topic><topic>DNA Repair - physiology</topic><topic>DNA, Bacterial</topic><topic>Escherichia coli - enzymology</topic><topic>Flavins - chemistry</topic><topic>Mutation</topic><topic>Protein Conformation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xu, Lei</creatorcontrib><creatorcontrib>Mu, Wanmeng</creatorcontrib><creatorcontrib>Ding, Yanwei</creatorcontrib><creatorcontrib>Luo, Zhaofeng</creatorcontrib><creatorcontrib>Han, Qingkai</creatorcontrib><creatorcontrib>Bi, Fuyong</creatorcontrib><creatorcontrib>Wang, Yuzhen</creatorcontrib><creatorcontrib>Song, Qinhua</creatorcontrib><collection>Istex</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>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xu, Lei</au><au>Mu, Wanmeng</au><au>Ding, Yanwei</au><au>Luo, Zhaofeng</au><au>Han, Qingkai</au><au>Bi, Fuyong</au><au>Wang, Yuzhen</au><au>Song, Qinhua</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2008-08-19</date><risdate>2008</risdate><volume>47</volume><issue>33</issue><spage>8736</spage><epage>8743</epage><pages>8736-8743</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH−). After purification in vitro, the cofactor FADH− in photolyase is oxidized into the neutral radical form FADH• under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FADox) but not FADH• after routine purification procedures, but evidence shows that the mutant protein contains FADH− in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>18652481</pmid><doi>10.1021/bi800391j</doi><tpages>8</tpages></addata></record> |
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| source | MEDLINE; ACS Publications |
| subjects | Asparagine - chemistry Binding Sites Deoxyribodipyrimidine Photo-Lyase - chemistry Deoxyribodipyrimidine Photo-Lyase - metabolism DNA Damage DNA Repair - physiology DNA, Bacterial Escherichia coli - enzymology Flavins - chemistry Mutation Protein Conformation |
| title | Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair |
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