Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis
The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of...
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| Veröffentlicht in: | Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2011, Vol.75 (11), p.2184-2193 |
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| container_title | Bioscience, biotechnology, and biochemistry |
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| creator | ASAMIZU, Shumpei SHIRO, Yoshitsugu IGARASHI, Yasuhiro NAGANO, Shingo ONAKA, Hiroyasu |
| description | The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively. |
| doi_str_mv | 10.1271/bbb.110474 |
| format | Article |
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In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively.</description><identifier>ISSN: 0916-8451</identifier><identifier>ISSN: 1347-6947</identifier><identifier>EISSN: 1347-6947</identifier><identifier>DOI: 10.1271/bbb.110474</identifier><identifier>PMID: 22056432</identifier><language>eng</language><publisher>Tokyo: Japan Society for Bioscience, Biotechnology, and Agrochemistry</publisher><subject>Amino Acid Sequence ; Binding Sites ; Biochemistry ; Biological and medical sciences ; Biotechnology ; Carbazoles - chemical synthesis ; Carbazoles - chemistry ; Carbazoles - metabolism ; Catalytic activity ; Cloning, Molecular ; Enzymes ; FAD-dependent monooxygenase ; Flavin-Adenine Dinucleotide - metabolism ; Fundamental and applied biological sciences. Psychology ; Glutamine ; Indole Alkaloids - chemistry ; Indole Alkaloids - metabolism ; Indoles - chemistry ; Indoles - metabolism ; indolocarbazole biosynthesis ; Molecular Sequence Data ; Molecular Structure ; mutational analysis ; Mutations ; Oxidation-Reduction ; Oxygenases - genetics ; Oxygenases - metabolism ; Proteins ; Pyrroles - chemistry ; secondary metabolism ; Staurosporine - genetics ; Staurosporine - metabolism ; Streptomyces ; Streptomyces - enzymology ; Streptomyces - genetics ; Substrate Specificity ; Valence</subject><ispartof>Bioscience, biotechnology, and biochemistry, 2011, Vol.75 (11), p.2184-2193</ispartof><rights>2011 by Japan Society for Bioscience, Biotechnology, and Agrochemistry 2011</rights><rights>2015 INIST-CNRS</rights><rights>Copyright Japan Science and Technology Agency 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c685t-74b2356e6fcd6bb5e01260f137c20e1021e67a12ea2b3ba7074d4afb120a727f3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,4009,27902,27903,27904</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=25472241$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22056432$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>ASAMIZU, Shumpei</creatorcontrib><creatorcontrib>SHIRO, Yoshitsugu</creatorcontrib><creatorcontrib>IGARASHI, Yasuhiro</creatorcontrib><creatorcontrib>NAGANO, Shingo</creatorcontrib><creatorcontrib>ONAKA, Hiroyasu</creatorcontrib><title>Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis</title><title>Bioscience, biotechnology, and biochemistry</title><addtitle>Biosci Biotechnol Biochem</addtitle><description>The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively.</description><subject>Amino Acid Sequence</subject><subject>Binding Sites</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Carbazoles - chemical synthesis</subject><subject>Carbazoles - chemistry</subject><subject>Carbazoles - metabolism</subject><subject>Catalytic activity</subject><subject>Cloning, Molecular</subject><subject>Enzymes</subject><subject>FAD-dependent monooxygenase</subject><subject>Flavin-Adenine Dinucleotide - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glutamine</subject><subject>Indole Alkaloids - chemistry</subject><subject>Indole Alkaloids - metabolism</subject><subject>Indoles - chemistry</subject><subject>Indoles - metabolism</subject><subject>indolocarbazole biosynthesis</subject><subject>Molecular Sequence Data</subject><subject>Molecular Structure</subject><subject>mutational analysis</subject><subject>Mutations</subject><subject>Oxidation-Reduction</subject><subject>Oxygenases - genetics</subject><subject>Oxygenases - metabolism</subject><subject>Proteins</subject><subject>Pyrroles - chemistry</subject><subject>secondary metabolism</subject><subject>Staurosporine - genetics</subject><subject>Staurosporine - metabolism</subject><subject>Streptomyces</subject><subject>Streptomyces - enzymology</subject><subject>Streptomyces - genetics</subject><subject>Substrate Specificity</subject><subject>Valence</subject><issn>0916-8451</issn><issn>1347-6947</issn><issn>1347-6947</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0d9r1TAUB_AiirtOX_wDJCCiiJ1Jmibt4yxOL0wm_sDHcpIm3IzcZCbt9O5P8K82tXcTRPQpJOdzwuF8i-IhwUeECvJSSnlECGaC3SpWpGKi5C0Tt4sVbgkvG1aTg-JeSucY54ea3C0OKMU1ZxVdFT-6DURQo472CkYbPAI_oJPJq_kCDr0LgzVWLbVg0McRul_mg5bdC_RlY9UGHUeN1v4yuEs9IOvRuNHo_S7G4DQ6-26Hm-61H4ILCqKEq7n4yoa085knm-4Xdwy4pB_sz8Pi88nrT93b8vTszbo7Pi0Vb-qxFEzSquaaGzVwKWuNCeXYkEooijXBlGgugFANVFYSBBZsYGAkoRgEFaY6LJ4u_17E8HXSaey3NintHHgdptS3pMG8pUz8X2Ke99niWT77pyRcEFY3rCKZPv6Dnocp5lVnxVjbMN42PKvni1IxpBS16S-i3ULc9QT3c-p9Tr1fUs_40f7LSW71cEOvY87gyR5AUuBMBK9s-u1qJihl82z14qw3IW7hW4hu6EfYuRCvm6q_DPATxo7GHg</recordid><startdate>2011</startdate><enddate>2011</enddate><creator>ASAMIZU, Shumpei</creator><creator>SHIRO, Yoshitsugu</creator><creator>IGARASHI, Yasuhiro</creator><creator>NAGANO, Shingo</creator><creator>ONAKA, Hiroyasu</creator><general>Japan Society for Bioscience, Biotechnology, and Agrochemistry</general><general>Japan Society for Bioscience Biotechnology and Agrochemistry</general><general>Oxford University Press</general><scope>IQODW</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>7TB</scope><scope>7U5</scope><scope>8FD</scope><scope>FR3</scope><scope>L7M</scope><scope>7X8</scope><scope>7QO</scope><scope>P64</scope></search><sort><creationdate>2011</creationdate><title>Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis</title><author>ASAMIZU, Shumpei ; SHIRO, Yoshitsugu ; IGARASHI, Yasuhiro ; NAGANO, Shingo ; ONAKA, Hiroyasu</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c685t-74b2356e6fcd6bb5e01260f137c20e1021e67a12ea2b3ba7074d4afb120a727f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Amino Acid Sequence</topic><topic>Binding Sites</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Carbazoles - chemical synthesis</topic><topic>Carbazoles - chemistry</topic><topic>Carbazoles - metabolism</topic><topic>Catalytic activity</topic><topic>Cloning, Molecular</topic><topic>Enzymes</topic><topic>FAD-dependent monooxygenase</topic><topic>Flavin-Adenine Dinucleotide - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glutamine</topic><topic>Indole Alkaloids - chemistry</topic><topic>Indole Alkaloids - metabolism</topic><topic>Indoles - chemistry</topic><topic>Indoles - metabolism</topic><topic>indolocarbazole biosynthesis</topic><topic>Molecular Sequence Data</topic><topic>Molecular Structure</topic><topic>mutational analysis</topic><topic>Mutations</topic><topic>Oxidation-Reduction</topic><topic>Oxygenases - genetics</topic><topic>Oxygenases - metabolism</topic><topic>Proteins</topic><topic>Pyrroles - chemistry</topic><topic>secondary metabolism</topic><topic>Staurosporine - genetics</topic><topic>Staurosporine - metabolism</topic><topic>Streptomyces</topic><topic>Streptomyces - enzymology</topic><topic>Streptomyces - genetics</topic><topic>Substrate Specificity</topic><topic>Valence</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>ASAMIZU, Shumpei</creatorcontrib><creatorcontrib>SHIRO, Yoshitsugu</creatorcontrib><creatorcontrib>IGARASHI, Yasuhiro</creatorcontrib><creatorcontrib>NAGANO, Shingo</creatorcontrib><creatorcontrib>ONAKA, Hiroyasu</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Bioscience, biotechnology, and biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>ASAMIZU, Shumpei</au><au>SHIRO, Yoshitsugu</au><au>IGARASHI, Yasuhiro</au><au>NAGANO, Shingo</au><au>ONAKA, Hiroyasu</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis</atitle><jtitle>Bioscience, biotechnology, and biochemistry</jtitle><addtitle>Biosci Biotechnol Biochem</addtitle><date>2011</date><risdate>2011</risdate><volume>75</volume><issue>11</issue><spage>2184</spage><epage>2193</epage><pages>2184-2193</pages><issn>0916-8451</issn><issn>1347-6947</issn><eissn>1347-6947</eissn><abstract>The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively.</abstract><cop>Tokyo</cop><pub>Japan Society for Bioscience, Biotechnology, and Agrochemistry</pub><pmid>22056432</pmid><doi>10.1271/bbb.110474</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Bioscience, biotechnology, and biochemistry, 2011, Vol.75 (11), p.2184-2193 |
| issn | 0916-8451 1347-6947 1347-6947 |
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| source | J-STAGE Free; MEDLINE; Oxford University Press Journals All Titles (1996-Current); Freely Accessible Japanese Titles; EZB-FREE-00999 freely available EZB journals; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence Binding Sites Biochemistry Biological and medical sciences Biotechnology Carbazoles - chemical synthesis Carbazoles - chemistry Carbazoles - metabolism Catalytic activity Cloning, Molecular Enzymes FAD-dependent monooxygenase Flavin-Adenine Dinucleotide - metabolism Fundamental and applied biological sciences. Psychology Glutamine Indole Alkaloids - chemistry Indole Alkaloids - metabolism Indoles - chemistry Indoles - metabolism indolocarbazole biosynthesis Molecular Sequence Data Molecular Structure mutational analysis Mutations Oxidation-Reduction Oxygenases - genetics Oxygenases - metabolism Proteins Pyrroles - chemistry secondary metabolism Staurosporine - genetics Staurosporine - metabolism Streptomyces Streptomyces - enzymology Streptomyces - genetics Substrate Specificity Valence |
| title | Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis |
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