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dc.contributor.authorKhandokar, Yogesh B
dc.contributor.authorSrivastava, Parul
dc.contributor.authorSarker, Subir
dc.contributor.authorSwarbrick, Crystall MD
dc.contributor.authorAragao, David
dc.contributor.authorCowieson, Nathan
dc.contributor.authorForwood, Jade K
dc.date.accessioned2020-02-13T23:54:44Z
dc.date.available2020-02-13T23:54:44Z
dc.date.issued2016
dc.identifier.issn0021-9258
dc.identifier.doi10.1074/jbc.M115.677484
dc.identifier.urihttp://hdl.handle.net/10072/391438
dc.description.abstractPaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn37, Asp52, and Thr68 are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr38-Tyr39. This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.
dc.description.peerreviewedYes
dc.languageEnglish
dc.language.isoeng
dc.publisherAmerican Society for Biochemistry and Molecular Biology
dc.relation.ispartofpagefrom1866
dc.relation.ispartofpageto1876
dc.relation.ispartofissue4
dc.relation.ispartofjournalJournal of Biological Chemistry
dc.relation.ispartofvolume291
dc.subject.fieldofresearchChemical Sciences
dc.subject.fieldofresearchBiological Sciences
dc.subject.fieldofresearchMedical and Health Sciences
dc.subject.fieldofresearchcode03
dc.subject.fieldofresearchcode06
dc.subject.fieldofresearchcode11
dc.subject.keywordsScience & Technology
dc.subject.keywordsLife Sciences & Biomedicine
dc.subject.keywordsBiochemistry & Molecular Biology
dc.subject.keywordsNEGATIVE COOPERATIVITY
dc.subject.keywordsCATALYTIC MECHANISM
dc.titleStructural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism
dc.typeJournal article
dc.type.descriptionC1 - Articles
dcterms.bibliographicCitationKhandokar, YB; Srivastava, P; Sarker, S; Swarbrick, CMD; Aragao, D; Cowieson, N; Forwood, JK, Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism, Journal of Biological Chemistry, 2016, 291 (4), pp. 1866-1876
dc.date.updated2020-02-13T23:50:44Z
dc.description.versionVersion of Record (VoR)
gro.rights.copyrightThis research was originally published in Journal of Biological Chemistry (JBC). Khandokar, et al., Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism, Journal of Biological Chemistry (JBC), 291, 1866-1876, 2016.. Copyright the American Society for Biochemistry and Molecular Biology. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive version.
gro.hasfulltextFull Text
gro.griffith.authorSwarbrick, Crystall


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