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Article: Probing the active site of mitogillin, a fungal ribotoxin

TitleProbing the active site of mitogillin, a fungal ribotoxin
Authors
Issue Date1998
PublisherBlackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journals/MMI
Citation
Molecular Microbiology, 1998, v. 29 n. 4, p. 1019-1027 How to Cite?
AbstractFungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14- base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.
Persistent Identifierhttp://hdl.handle.net/10722/157286
ISSN
2015 Impact Factor: 3.761
2015 SCImago Journal Rankings: 2.978
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorKao, Ren_US
dc.contributor.authorShea, JEen_US
dc.contributor.authorDavies, Jen_US
dc.contributor.authorHolden, DWen_US
dc.date.accessioned2012-08-08T08:48:40Z-
dc.date.available2012-08-08T08:48:40Z-
dc.date.issued1998en_US
dc.identifier.citationMolecular Microbiology, 1998, v. 29 n. 4, p. 1019-1027en_US
dc.identifier.issn0950-382Xen_US
dc.identifier.urihttp://hdl.handle.net/10722/157286-
dc.description.abstractFungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14- base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.en_US
dc.languageengen_US
dc.publisherBlackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journals/MMIen_US
dc.relation.ispartofMolecular Microbiologyen_US
dc.subject.meshAllergensen_US
dc.subject.meshAnimalsen_US
dc.subject.meshAntigens, Planten_US
dc.subject.meshAspergillus Fumigatus - Geneticsen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshBinding Sites - Geneticsen_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshDna Primers - Geneticsen_US
dc.subject.meshEscherichia Coli - Geneticsen_US
dc.subject.meshFungal Proteins - Genetics - Metabolism - Toxicityen_US
dc.subject.meshGenes, Fungalen_US
dc.subject.meshGenetic Vectorsen_US
dc.subject.meshHydroxylamineen_US
dc.subject.meshMutagensen_US
dc.subject.meshMycotoxins - Genetics - Metabolism - Toxicityen_US
dc.subject.meshPlasmids - Geneticsen_US
dc.subject.meshPoint Mutationen_US
dc.subject.meshRabbitsen_US
dc.subject.meshReticulocytes - Drug Effects - Metabolismen_US
dc.subject.meshRibonucleases - Genetics - Metabolismen_US
dc.subject.meshSaccharomyces Cerevisiae - Geneticsen_US
dc.titleProbing the active site of mitogillin, a fungal ribotoxinen_US
dc.typeArticleen_US
dc.identifier.emailKao, R:rytkao@hkucc.hku.hken_US
dc.identifier.authorityKao, R=rp00481en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1046/j.1365-2958.1998.00987.xen_US
dc.identifier.pmid9767570en_US
dc.identifier.scopuseid_2-s2.0-0031816245en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0031816245&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume29en_US
dc.identifier.issue4en_US
dc.identifier.spage1019en_US
dc.identifier.epage1027en_US
dc.identifier.isiWOS:000075451700009-
dc.publisher.placeUnited Kingdomen_US
dc.identifier.scopusauthoridKao, R=7101675499en_US
dc.identifier.scopusauthoridShea, JE=7202997189en_US
dc.identifier.scopusauthoridDavies, J=7404982789en_US
dc.identifier.scopusauthoridHolden, DW=34770112500en_US

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