Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954
Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combin...
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description | Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme–prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an
E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied. |
doi_str_mv | 10.1016/j.jmb.2007.02.012 |
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E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/j.jmb.2007.02.012</identifier><identifier>PMID: 17350040</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>Aziridines - chemistry ; Aziridines - metabolism ; Binding Sites ; Catalysis ; CB1954 ; Chromatography, High Pressure Liquid ; Crystallography, X-Ray ; Escherichia coli ; Escherichia coli - enzymology ; gene therapy ; Hydroxylamine ; Kinetics ; Models, Molecular ; Mutant Proteins - chemistry ; Mutation - genetics ; Niacin - metabolism ; Nitrofurazone - metabolism ; nitroreductase ; Nitroreductases - chemistry ; Oxidation-Reduction ; Prodrugs - metabolism ; protein engineering ; Protein Structure, Secondary ; Substrate Specificity ; X-ray structure</subject><ispartof>Journal of molecular biology, 2007-04, Vol.368 (2), p.481-492</ispartof><rights>2007 Elsevier Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c382t-5c0d1f9973e9b1f4edc907692781d6e26357902dc6b2632602c4426c0e61c0bb3</citedby><cites>FETCH-LOGICAL-c382t-5c0d1f9973e9b1f4edc907692781d6e26357902dc6b2632602c4426c0e61c0bb3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jmb.2007.02.012$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17350040$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Race, Paul R.</creatorcontrib><creatorcontrib>Lovering, Andrew L.</creatorcontrib><creatorcontrib>White, Scott A.</creatorcontrib><creatorcontrib>Grove, Jane I.</creatorcontrib><creatorcontrib>Searle, Peter F.</creatorcontrib><creatorcontrib>Wrighton, Christopher W.</creatorcontrib><creatorcontrib>Hyde, EvaI</creatorcontrib><title>Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><description>Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme–prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an
E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.</description><subject>Aziridines - chemistry</subject><subject>Aziridines - metabolism</subject><subject>Binding Sites</subject><subject>Catalysis</subject><subject>CB1954</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Crystallography, X-Ray</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>gene therapy</subject><subject>Hydroxylamine</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Mutant Proteins - chemistry</subject><subject>Mutation - genetics</subject><subject>Niacin - metabolism</subject><subject>Nitrofurazone - metabolism</subject><subject>nitroreductase</subject><subject>Nitroreductases - chemistry</subject><subject>Oxidation-Reduction</subject><subject>Prodrugs - metabolism</subject><subject>protein engineering</subject><subject>Protein Structure, Secondary</subject><subject>Substrate Specificity</subject><subject>X-ray structure</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcuO1DAQRS0EYpqBD2CDvGKXULbzFCto9cCI4SE1rC2nXJm4lcSD7Qyaj-CfSatbYgerskrnXpV8GHspIBcgqjeH_DB1uQSoc5A5CPmIbQQ0bdZUqnnMNgBSZrJR1QV7FuMBAEpVNE_ZhahVCVDAhv3-5GZKDrmZLd-nsGBaghn5djDBYKLgoknOz9z3fBdxWBc4OMPRj45_cSn4QHYNmUj885LMnCLfD_6Xm2_59XQX_D1Zvut7hwYfeO8DTwPxb8HbsNzy_dLFFEwivn0v2rJ4zp70Zoz04jwv2Y-r3fftx-zm64fr7bubDFUjU1YiWNG3ba2o7URfkMUW6qqVdSNsRbJSZd2CtFh161tWILEoZIVAlUDoOnXJXp961wN_LhSTnlxEGkczk1-irkGpRqnyv-DxaNnCERQnEIOPMVCv74KbTHjQAvRRlj7oVZY-ytIg9Sprzbw6ly_dRPZv4mxnBd6eAFr_4t5R0BEdzUjWBcKkrXf_qP8DvQ2lnA</recordid><startdate>20070427</startdate><enddate>20070427</enddate><creator>Race, Paul R.</creator><creator>Lovering, Andrew L.</creator><creator>White, Scott A.</creator><creator>Grove, Jane I.</creator><creator>Searle, Peter F.</creator><creator>Wrighton, Christopher W.</creator><creator>Hyde, EvaI</creator><general>Elsevier Ltd</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>7U9</scope><scope>C1K</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20070427</creationdate><title>Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954</title><author>Race, Paul R. ; Lovering, Andrew L. ; White, Scott A. ; Grove, Jane I. ; Searle, Peter F. ; Wrighton, Christopher W. ; Hyde, EvaI</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c382t-5c0d1f9973e9b1f4edc907692781d6e26357902dc6b2632602c4426c0e61c0bb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Aziridines - chemistry</topic><topic>Aziridines - metabolism</topic><topic>Binding Sites</topic><topic>Catalysis</topic><topic>CB1954</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Crystallography, X-Ray</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>gene therapy</topic><topic>Hydroxylamine</topic><topic>Kinetics</topic><topic>Models, Molecular</topic><topic>Mutant Proteins - chemistry</topic><topic>Mutation - genetics</topic><topic>Niacin - metabolism</topic><topic>Nitrofurazone - metabolism</topic><topic>nitroreductase</topic><topic>Nitroreductases - chemistry</topic><topic>Oxidation-Reduction</topic><topic>Prodrugs - metabolism</topic><topic>protein engineering</topic><topic>Protein Structure, Secondary</topic><topic>Substrate Specificity</topic><topic>X-ray structure</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Race, Paul R.</creatorcontrib><creatorcontrib>Lovering, Andrew L.</creatorcontrib><creatorcontrib>White, Scott A.</creatorcontrib><creatorcontrib>Grove, Jane I.</creatorcontrib><creatorcontrib>Searle, Peter F.</creatorcontrib><creatorcontrib>Wrighton, Christopher W.</creatorcontrib><creatorcontrib>Hyde, EvaI</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>Virology and AIDS Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Race, Paul R.</au><au>Lovering, Andrew L.</au><au>White, Scott A.</au><au>Grove, Jane I.</au><au>Searle, Peter F.</au><au>Wrighton, Christopher W.</au><au>Hyde, EvaI</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>2007-04-27</date><risdate>2007</risdate><volume>368</volume><issue>2</issue><spage>481</spage><epage>492</epage><pages>481-492</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><abstract>Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme–prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an
E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>17350040</pmid><doi>10.1016/j.jmb.2007.02.012</doi><tpages>12</tpages></addata></record> |
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subjects | Aziridines - chemistry Aziridines - metabolism Binding Sites Catalysis CB1954 Chromatography, High Pressure Liquid Crystallography, X-Ray Escherichia coli Escherichia coli - enzymology gene therapy Hydroxylamine Kinetics Models, Molecular Mutant Proteins - chemistry Mutation - genetics Niacin - metabolism Nitrofurazone - metabolism nitroreductase Nitroreductases - chemistry Oxidation-Reduction Prodrugs - metabolism protein engineering Protein Structure, Secondary Substrate Specificity X-ray structure |
title | Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954 |
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