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Article: Fluorophore-labeled β-lactamase as a biosensor for β-lactam antibiotics: A study of the biosensing process

TitleFluorophore-labeled β-lactamase as a biosensor for β-lactam antibiotics: A study of the biosensing process
Authors
Issue Date2008
PublisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/journals/jacsat/index.html
Citation
Journal Of The American Chemical Society, 2008, v. 130 n. 20, p. 6351-6361 How to Cite?
AbstractThe fluorescein-labeled E166C mutant of the PenPC β-lactamase (E166Cf) represents a successful model in the construction of "switch-on" fluorescent biosensors from nonallosteric proteins (Chan P.-H. et al.; J. Am Chem. Soc, 2004, 126, 4074). This paper focuses on the study of the biosensing mechanism by which the E166Cf biosensor changes its fluorescence upon β-lactam binding and hydrolysis. Mass spectrometric and stopped-flow fluorescence studies of E166Cf with cefuroxime, penicillin G, and 6-aminopenicillanic acid reveal that the formation of enzyme-substrate complex enhances the fluorescence of E166Cf, and the subsequent regeneration of the free enzyme restores the weak fluorescence of E166Cf. Molecular modeling studies of E166Cf with penicillin G show that the fluorescein label is likely to share a common space with the β-lactam and thiazolidine rings of the antibiotic in the active site. This spatial clash appears to cause the fluorescein label to move from the active site to the external aqueous environment upon substrate binding and hence experience higher water exposure. Steady-state fluorescence measurements indicate that the fluorescence of E166Cf can be enhanced by 6-aminopenicillanic acid, which consists of the β-lactam and thiazolidine rings only. Thermal denaturation experiments of the wild-type enzyme, E166C, and E166Cf reveal that the E166C mutation is likely to increase the flexibility of the Ω-loop. This "modified" structural property might compensate for the possible steric effect of the fluorescein label on substrate binding. © 2008 American Chemical Society.
Persistent Identifierhttp://hdl.handle.net/10722/155906
ISSN
2015 Impact Factor: 13.038
2015 SCImago Journal Rankings: 7.123
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorChan, PHen_HK
dc.contributor.authorSo, PKen_HK
dc.contributor.authorMa, DLen_HK
dc.contributor.authorZhao, Yen_HK
dc.contributor.authorLai, TSen_HK
dc.contributor.authorChung, WHen_HK
dc.contributor.authorChan, KCen_HK
dc.contributor.authorYiu, KFen_HK
dc.contributor.authorChan, HWen_HK
dc.contributor.authorSiu, FMen_HK
dc.contributor.authorTsang, CWen_HK
dc.contributor.authorLeung, YCen_HK
dc.contributor.authorWong, KYen_HK
dc.date.accessioned2012-08-08T08:38:19Z-
dc.date.available2012-08-08T08:38:19Z-
dc.date.issued2008en_HK
dc.identifier.citationJournal Of The American Chemical Society, 2008, v. 130 n. 20, p. 6351-6361en_HK
dc.identifier.issn0002-7863en_HK
dc.identifier.urihttp://hdl.handle.net/10722/155906-
dc.description.abstractThe fluorescein-labeled E166C mutant of the PenPC β-lactamase (E166Cf) represents a successful model in the construction of "switch-on" fluorescent biosensors from nonallosteric proteins (Chan P.-H. et al.; J. Am Chem. Soc, 2004, 126, 4074). This paper focuses on the study of the biosensing mechanism by which the E166Cf biosensor changes its fluorescence upon β-lactam binding and hydrolysis. Mass spectrometric and stopped-flow fluorescence studies of E166Cf with cefuroxime, penicillin G, and 6-aminopenicillanic acid reveal that the formation of enzyme-substrate complex enhances the fluorescence of E166Cf, and the subsequent regeneration of the free enzyme restores the weak fluorescence of E166Cf. Molecular modeling studies of E166Cf with penicillin G show that the fluorescein label is likely to share a common space with the β-lactam and thiazolidine rings of the antibiotic in the active site. This spatial clash appears to cause the fluorescein label to move from the active site to the external aqueous environment upon substrate binding and hence experience higher water exposure. Steady-state fluorescence measurements indicate that the fluorescence of E166Cf can be enhanced by 6-aminopenicillanic acid, which consists of the β-lactam and thiazolidine rings only. Thermal denaturation experiments of the wild-type enzyme, E166C, and E166Cf reveal that the E166C mutation is likely to increase the flexibility of the Ω-loop. This "modified" structural property might compensate for the possible steric effect of the fluorescein label on substrate binding. © 2008 American Chemical Society.en_HK
dc.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/journals/jacsat/index.htmlen_HK
dc.relation.ispartofJournal of the American Chemical Societyen_HK
dc.subject.meshAnti-Bacterial Agents - Analysis - Chemistryen_US
dc.subject.meshBiosensing Techniques - Methodsen_US
dc.subject.meshCefuroxime - Analysis - Chemistryen_US
dc.subject.meshCircular Dichroismen_US
dc.subject.meshComputer Simulationen_US
dc.subject.meshFluoresceins - Chemistryen_US
dc.subject.meshFluorescent Dyes - Chemistryen_US
dc.subject.meshKineticsen_US
dc.subject.meshMass Spectrometryen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshPenicillanic Acid - Analogs & Derivatives - Analysis - Chemistryen_US
dc.subject.meshPenicillin G - Analysis - Chemistryen_US
dc.subject.meshProtein Denaturationen_US
dc.subject.meshSpectrometry, Fluorescence - Methodsen_US
dc.subject.meshBeta-Lactamases - Chemistry - Metabolismen_US
dc.subject.meshBeta-Lactams - Analysis - Chemistryen_US
dc.titleFluorophore-labeled β-lactamase as a biosensor for β-lactam antibiotics: A study of the biosensing processen_HK
dc.typeArticleen_HK
dc.identifier.emailMa, DL:edmondma@hku.hken_HK
dc.identifier.emailYiu, KF:cedric@hkucc.hku.hken_HK
dc.identifier.emailSiu, FM:fmsiu@hku.hken_HK
dc.identifier.authorityMa, DL=rp00760en_HK
dc.identifier.authorityYiu, KF=rp00206en_HK
dc.identifier.authoritySiu, FM=rp00776en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1021/ja076111gen_HK
dc.identifier.pmid18429614-
dc.identifier.scopuseid_2-s2.0-43949099291en_HK
dc.identifier.hkuros142667-
dc.identifier.hkuros155846-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-43949099291&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume130en_HK
dc.identifier.issue20en_HK
dc.identifier.spage6351en_HK
dc.identifier.epage6361en_HK
dc.identifier.isiWOS:000255854100024-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridChan, PH=36545749700en_HK
dc.identifier.scopusauthoridSo, PK=14919747700en_HK
dc.identifier.scopusauthoridMa, DL=7402075538en_HK
dc.identifier.scopusauthoridZhao, Y=7407398716en_HK
dc.identifier.scopusauthoridLai, TS=7202203490en_HK
dc.identifier.scopusauthoridChung, WH=8722115800en_HK
dc.identifier.scopusauthoridChan, KC=24444179400en_HK
dc.identifier.scopusauthoridYiu, KF=24802813000en_HK
dc.identifier.scopusauthoridChan, HW=36842410300en_HK
dc.identifier.scopusauthoridSiu, FM=6701518489en_HK
dc.identifier.scopusauthoridTsang, CW=7202935952en_HK
dc.identifier.scopusauthoridLeung, YC=35074432700en_HK
dc.identifier.scopusauthoridWong, KY=26641515400en_HK

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