Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease
UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease...
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| description | UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented. |
| doi_str_mv | 10.1371/journal.pone.0032918 |
| format | Article |
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Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0032918</identifier><identifier>PMID: 22448231</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Anisotropy ; Biochemistry ; Biology ; Catalysis ; Chagas Disease ; Chemical kinetics ; Chemistry ; Chromatography ; Drug development ; Drugs ; Enzymes ; Flavin ; Flavins - metabolism ; Fluorescence ; Galactose ; Galactose - analogs & derivatives ; Galactose - metabolism ; Humans ; Intermediates ; Intramolecular Transferases - chemistry ; Intramolecular Transferases - metabolism ; Kinetics ; Laboratories ; Molecular Structure ; Mutagenesis ; NADP ; NADP - metabolism ; Oxidation-Reduction ; Oxygen ; Parasites ; Protein Conformation ; Protein expression ; R&D ; Reaction kinetics ; Redox reactions ; Research & development ; Substrate Specificity ; Trypanosoma cruzi ; Trypanosoma cruzi - enzymology ; Uridine Diphosphate - analogs & derivatives ; Uridine Diphosphate - metabolism ; Vector-borne diseases ; Virulence ; Virulence (Microbiology)</subject><ispartof>PloS one, 2012-03, Vol.7 (3), p.e32918-e32918</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 Oppenheimer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Oppenheimer et al. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c691t-d46edf10989e520b6c186c4349811789081edbeb1254bd738d153367cf9a74673</citedby><cites>FETCH-LOGICAL-c691t-d46edf10989e520b6c186c4349811789081edbeb1254bd738d153367cf9a74673</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308961/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308961/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,725,778,782,862,883,2098,2917,23853,27911,27912,53778,53780,79355,79356</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22448231$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Mariño-Ramírez, Leonardo</contributor><creatorcontrib>Oppenheimer, Michelle</creatorcontrib><creatorcontrib>Valenciano, Ana Lisa</creatorcontrib><creatorcontrib>Kizjakina, Karina</creatorcontrib><creatorcontrib>Qi, Jun</creatorcontrib><creatorcontrib>Sobrado, Pablo</creatorcontrib><title>Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.</description><subject>Anisotropy</subject><subject>Biochemistry</subject><subject>Biology</subject><subject>Catalysis</subject><subject>Chagas Disease</subject><subject>Chemical kinetics</subject><subject>Chemistry</subject><subject>Chromatography</subject><subject>Drug development</subject><subject>Drugs</subject><subject>Enzymes</subject><subject>Flavin</subject><subject>Flavins - metabolism</subject><subject>Fluorescence</subject><subject>Galactose</subject><subject>Galactose - analogs & derivatives</subject><subject>Galactose - metabolism</subject><subject>Humans</subject><subject>Intermediates</subject><subject>Intramolecular Transferases - chemistry</subject><subject>Intramolecular Transferases - metabolism</subject><subject>Kinetics</subject><subject>Laboratories</subject><subject>Molecular Structure</subject><subject>Mutagenesis</subject><subject>NADP</subject><subject>NADP - metabolism</subject><subject>Oxidation-Reduction</subject><subject>Oxygen</subject><subject>Parasites</subject><subject>Protein Conformation</subject><subject>Protein expression</subject><subject>R&D</subject><subject>Reaction kinetics</subject><subject>Redox reactions</subject><subject>Research & development</subject><subject>Substrate Specificity</subject><subject>Trypanosoma cruzi</subject><subject>Trypanosoma cruzi - enzymology</subject><subject>Uridine Diphosphate - analogs & derivatives</subject><subject>Uridine Diphosphate - metabolism</subject><subject>Vector-borne diseases</subject><subject>Virulence</subject><subject>Virulence (Microbiology)</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk12P1CAUhhujcdfRf2CUxMSNFzNCobR4YbIZvybZZI3uekso0A6TtlSgZsdfL3W6m6nZC8MF5PCc98ALJ0meI7hCOEdvd3ZwnWhWve30CkKcMlQ8SE4Rw-mSphA_PFqfJE-830GY4YLSx8lJmhJSpBidJjfrrW6NFA1otdyKzvgW2Apcf_i6rEUjZLD93onOeg3aIYg4Vc624Mrt-zFqWwGkG36bd0CA3gbdBRO1lBtqEISrdQCiFqbzAay3ceXPgDJeR52nyaNKNF4_m-ZFcv3p49X6y_Li8vNmfX6xlJShsFSEalUhyAqmsxSWVKKCSoIJKxDKCwYLpFWpS5RmpFQ5LhTKMKa5rJjICc3xInl50O0b6_lkmucIRwtwhiK9SDYHQlmx470zrXB7boXhfwPW1Vy4YGSjeTxLRiCqqNI5yZVgpII5KVMsEcuoGqu9n6oNZauVjH440cxE5zud2fLa_uIYw4JRFAXOJgFnfw7aB94aL3XTiE7bwXNGChbPXuBIvvqHvP9yExVfU3PTVTaWlaMmPyd5DjNKszRSq3uoONT4OeIHq0yMzxLezBIiE_RNqMXgPd98__b_7OWPOfv6iN1q0YStt80QjO38HCQHUDrrvdPVnccI8rE_bt3gY3_wqT9i2ovj97lLum0I_AcJbQqF</recordid><startdate>20120320</startdate><enddate>20120320</enddate><creator>Oppenheimer, Michelle</creator><creator>Valenciano, Ana Lisa</creator><creator>Kizjakina, Karina</creator><creator>Qi, Jun</creator><creator>Sobrado, Pablo</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20120320</creationdate><title>Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease</title><author>Oppenheimer, Michelle ; Valenciano, Ana Lisa ; Kizjakina, Karina ; Qi, Jun ; Sobrado, Pablo</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c691t-d46edf10989e520b6c186c4349811789081edbeb1254bd738d153367cf9a74673</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Anisotropy</topic><topic>Biochemistry</topic><topic>Biology</topic><topic>Catalysis</topic><topic>Chagas Disease</topic><topic>Chemical kinetics</topic><topic>Chemistry</topic><topic>Chromatography</topic><topic>Drug development</topic><topic>Drugs</topic><topic>Enzymes</topic><topic>Flavin</topic><topic>Flavins - metabolism</topic><topic>Fluorescence</topic><topic>Galactose</topic><topic>Galactose - analogs & derivatives</topic><topic>Galactose - metabolism</topic><topic>Humans</topic><topic>Intermediates</topic><topic>Intramolecular Transferases - chemistry</topic><topic>Intramolecular Transferases - metabolism</topic><topic>Kinetics</topic><topic>Laboratories</topic><topic>Molecular Structure</topic><topic>Mutagenesis</topic><topic>NADP</topic><topic>NADP - metabolism</topic><topic>Oxidation-Reduction</topic><topic>Oxygen</topic><topic>Parasites</topic><topic>Protein Conformation</topic><topic>Protein expression</topic><topic>R&D</topic><topic>Reaction kinetics</topic><topic>Redox reactions</topic><topic>Research & development</topic><topic>Substrate Specificity</topic><topic>Trypanosoma cruzi</topic><topic>Trypanosoma cruzi - enzymology</topic><topic>Uridine Diphosphate - analogs & derivatives</topic><topic>Uridine Diphosphate - metabolism</topic><topic>Vector-borne diseases</topic><topic>Virulence</topic><topic>Virulence (Microbiology)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Oppenheimer, Michelle</creatorcontrib><creatorcontrib>Valenciano, Ana Lisa</creatorcontrib><creatorcontrib>Kizjakina, Karina</creatorcontrib><creatorcontrib>Qi, Jun</creatorcontrib><creatorcontrib>Sobrado, Pablo</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection (ProQuest)</collection><collection>Natural Science Collection (ProQuest)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Oppenheimer, Michelle</au><au>Valenciano, Ana Lisa</au><au>Kizjakina, Karina</au><au>Qi, Jun</au><au>Sobrado, Pablo</au><au>Mariño-Ramírez, Leonardo</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-03-20</date><risdate>2012</risdate><volume>7</volume><issue>3</issue><spage>e32918</spage><epage>e32918</epage><pages>e32918-e32918</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22448231</pmid><doi>10.1371/journal.pone.0032918</doi><tpages>e32918</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2012-03, Vol.7 (3), p.e32918-e32918 |
| issn | 1932-6203 1932-6203 |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Anisotropy Biochemistry Biology Catalysis Chagas Disease Chemical kinetics Chemistry Chromatography Drug development Drugs Enzymes Flavin Flavins - metabolism Fluorescence Galactose Galactose - analogs & derivatives Galactose - metabolism Humans Intermediates Intramolecular Transferases - chemistry Intramolecular Transferases - metabolism Kinetics Laboratories Molecular Structure Mutagenesis NADP NADP - metabolism Oxidation-Reduction Oxygen Parasites Protein Conformation Protein expression R&D Reaction kinetics Redox reactions Research & development Substrate Specificity Trypanosoma cruzi Trypanosoma cruzi - enzymology Uridine Diphosphate - analogs & derivatives Uridine Diphosphate - metabolism Vector-borne diseases Virulence Virulence (Microbiology) |
| title | Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-15T13%3A48%3A09IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Chemical%20mechanism%20of%20UDP-galactopyranose%20mutase%20from%20Trypanosoma%20cruzi:%20a%20potential%20drug%20target%20against%20Chagas'%20disease&rft.jtitle=PloS%20one&rft.au=Oppenheimer,%20Michelle&rft.date=2012-03-20&rft.volume=7&rft.issue=3&rft.spage=e32918&rft.epage=e32918&rft.pages=e32918-e32918&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0032918&rft_dat=%3Cgale_plos_%3EA477056652%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1324435115&rft_id=info:pmid/22448231&rft_galeid=A477056652&rft_doaj_id=oai_doaj_org_article_6ed5401f6de747da94f074b23c1956d7&rfr_iscdi=true |