Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain
Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited sta...
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description | Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866−2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved β-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent β-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions. |
doi_str_mv | 10.1021/bi047903f |
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The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866−2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved β-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent β-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi047903f</identifier><identifier>PMID: 15683224</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Adenosine Monophosphate - chemistry ; Amino Acid Motifs - genetics ; Amino Acid Substitution - genetics ; Animals ; Catalysis ; Coenzyme A - chemistry ; Fireflies - enzymology ; Kinetics ; Luciferases, Firefly - chemistry ; Luciferases, Firefly - genetics ; Luciferases, Firefly - isolation & purification ; Luminescence ; Models, Molecular ; Mutagenesis, Site-Directed ; Oxidation-Reduction ; Peptide Fragments - chemistry ; Peptide Fragments - genetics ; Peptide Fragments - isolation & purification ; Protein Conformation ; Protein Structure, Tertiary - genetics</subject><ispartof>Biochemistry (Easton), 2005-02, Vol.44 (5), p.1385-1393</ispartof><rights>Copyright © 2005 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a351t-f687da729860e8040e65bf64221536374c126c6286e8b2e6da3bbb0548e0aae73</citedby><cites>FETCH-LOGICAL-a351t-f687da729860e8040e65bf64221536374c126c6286e8b2e6da3bbb0548e0aae73</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/bi047903f$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi047903f$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15683224$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Branchini, Bruce R</creatorcontrib><creatorcontrib>Southworth, Tara L</creatorcontrib><creatorcontrib>Murtiashaw, Martha H</creatorcontrib><creatorcontrib>Wilkinson, Sara R</creatorcontrib><creatorcontrib>Khattak, Neelum F</creatorcontrib><creatorcontrib>Rosenberg, Justin C</creatorcontrib><creatorcontrib>Zimmer, Marc</creatorcontrib><title>Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866−2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved β-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent β-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.</description><subject>Adenosine Monophosphate - chemistry</subject><subject>Amino Acid Motifs - genetics</subject><subject>Amino Acid Substitution - genetics</subject><subject>Animals</subject><subject>Catalysis</subject><subject>Coenzyme A - chemistry</subject><subject>Fireflies - enzymology</subject><subject>Kinetics</subject><subject>Luciferases, Firefly - chemistry</subject><subject>Luciferases, Firefly - genetics</subject><subject>Luciferases, Firefly - isolation & purification</subject><subject>Luminescence</subject><subject>Models, Molecular</subject><subject>Mutagenesis, Site-Directed</subject><subject>Oxidation-Reduction</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - isolation & purification</subject><subject>Protein Conformation</subject><subject>Protein Structure, Tertiary - genetics</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkM1O3DAUha2qVRloF7xA5U0XLEJtJ7aTJR3-Kg2CwlSt2Fg3yXUx5AfZTsX0Rfq6GAZNN2x8Zd3vnKtzCNnlbJ8zwb_UjhW6Yrl9Q2ZcCpYVVSXfkhljTGWiUmyLbIdwm74F08V7ssWlKnMhihn5dzZF-I0DBhfo0R_X4tAgjTcQ04P0Anx00NFLhCa6cQh0tPTYebTdin51Yzf1LmmbZ9WBRzqHCN3qL7a0XtFDZy16HCKdj4MdfQ8bjyfzxdS4tIeQZNkSfbJKpw7HHtzwgbyz0AX8-DJ3yI_jo-X8NFucn3ybHywyyCWPmVWlbkGLqlQMyxQPlaytKoTgMle5LhouVKNEqbCsBaoW8rqumSxKZACo8x2yt_Zt_BhCymXuvevBrwxn5qlcsyk3sZ_W7P1U99j-J1_aTEC2BlyI-LDZg78zSudamuXFldHy-_XZ8qcwvxL_ec1DE8ztOPmUP7xy-BEFFpG6</recordid><startdate>20050208</startdate><enddate>20050208</enddate><creator>Branchini, Bruce R</creator><creator>Southworth, Tara L</creator><creator>Murtiashaw, Martha H</creator><creator>Wilkinson, Sara R</creator><creator>Khattak, Neelum F</creator><creator>Rosenberg, Justin C</creator><creator>Zimmer, Marc</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></search><sort><creationdate>20050208</creationdate><title>Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain</title><author>Branchini, Bruce R ; Southworth, Tara L ; Murtiashaw, Martha H ; Wilkinson, Sara R ; Khattak, Neelum F ; Rosenberg, Justin C ; Zimmer, Marc</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a351t-f687da729860e8040e65bf64221536374c126c6286e8b2e6da3bbb0548e0aae73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Adenosine Monophosphate - chemistry</topic><topic>Amino Acid Motifs - genetics</topic><topic>Amino Acid Substitution - genetics</topic><topic>Animals</topic><topic>Catalysis</topic><topic>Coenzyme A - chemistry</topic><topic>Fireflies - enzymology</topic><topic>Kinetics</topic><topic>Luciferases, Firefly - chemistry</topic><topic>Luciferases, Firefly - genetics</topic><topic>Luciferases, Firefly - isolation & purification</topic><topic>Luminescence</topic><topic>Models, Molecular</topic><topic>Mutagenesis, Site-Directed</topic><topic>Oxidation-Reduction</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - isolation & purification</topic><topic>Protein Conformation</topic><topic>Protein Structure, Tertiary - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Branchini, Bruce R</creatorcontrib><creatorcontrib>Southworth, Tara L</creatorcontrib><creatorcontrib>Murtiashaw, Martha H</creatorcontrib><creatorcontrib>Wilkinson, Sara R</creatorcontrib><creatorcontrib>Khattak, Neelum F</creatorcontrib><creatorcontrib>Rosenberg, Justin C</creatorcontrib><creatorcontrib>Zimmer, Marc</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><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Branchini, Bruce R</au><au>Southworth, Tara L</au><au>Murtiashaw, Martha H</au><au>Wilkinson, Sara R</au><au>Khattak, Neelum F</au><au>Rosenberg, Justin C</au><au>Zimmer, Marc</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2005-02-08</date><risdate>2005</risdate><volume>44</volume><issue>5</issue><spage>1385</spage><epage>1393</epage><pages>1385-1393</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866−2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved β-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent β-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>15683224</pmid><doi>10.1021/bi047903f</doi><tpages>9</tpages></addata></record> |
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subjects | Adenosine Monophosphate - chemistry Amino Acid Motifs - genetics Amino Acid Substitution - genetics Animals Catalysis Coenzyme A - chemistry Fireflies - enzymology Kinetics Luciferases, Firefly - chemistry Luciferases, Firefly - genetics Luciferases, Firefly - isolation & purification Luminescence Models, Molecular Mutagenesis, Site-Directed Oxidation-Reduction Peptide Fragments - chemistry Peptide Fragments - genetics Peptide Fragments - isolation & purification Protein Conformation Protein Structure, Tertiary - genetics |
title | Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain |
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