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Article: Tryptophanyl-tRNA synthetase urzyme: A model to recapitulate molecular evolution and investigate intramolecular complementation

TitleTryptophanyl-tRNA synthetase urzyme: A model to recapitulate molecular evolution and investigate intramolecular complementation
Authors
Issue Date2010
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 2010, v. 285 n. 49, p. 38590-38601 How to Cite?
AbstractWe substantiate our preliminary description of the class I tryptophanyl-tRNA synthetase minimal catalytic domain with details of its construction, structure, and steady-state kinetic parameters. Generating that active fragment involved deleting 65% of the contemporary enzyme, including the anticodon-binding domain and connecting peptide 1, CP1, a 74-residue internal segment from within the Rossmann fold. We used protein design (Rosetta), rather than phylogenetic sequence alignments, to identify mutations to compensate for the severe loss of modularity, thus restoring stability, as evidenced by renaturation described previously and by 70-ns molecular dynamics simulations. Sufficient solubility to enable biochemical studies was achieved by expressing the redesigned Urzyme as a maltose-binding protein fusion. Michaelis-Menten kinetic parameters from amino acid activation assays showed that, compared with the native full-length enzyme, TrpRS Urzyme binds ATP with similar affinity. This suggests that neither of the two deleted structural modules has a strong influence on ground-state ATP binding. However, tryptophan has 103 lower affinity, and the Urzyme has comparably reduced specificity relative to the related amino acid, tyrosine. Molecular dynamics simulations revealed how CP1 may contribute significantly to cognate amino acid specificity. As class Ia editing domains are nested within the CP1, this finding suggests that this module enhanced amino acid specificity continuously, throughout their evolution. We call this type of reconstructed protein catalyst an Urzyme (Ur prefix indicates original, primitive, or earliest). It establishes a model for recapitulating very early steps in molecular evolution in which fitness may have been enhanced by accumulating entire modules, rather than by discrete amino acid sequence changes. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Persistent Identifierhttp://hdl.handle.net/10722/142325
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
NIHGM078227
Funding Information:

This work is supported by NIH grant GM078227

References

 

DC FieldValueLanguage
dc.contributor.authorPham, Yen_HK
dc.contributor.authorKuhlman, Ben_HK
dc.contributor.authorButterfoss, GLen_HK
dc.contributor.authorHu, Hen_HK
dc.contributor.authorWeinreb, Ven_HK
dc.contributor.authorCarter Jr, CWen_HK
dc.date.accessioned2011-10-28T02:43:10Z-
dc.date.available2011-10-28T02:43:10Z-
dc.date.issued2010en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2010, v. 285 n. 49, p. 38590-38601en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/142325-
dc.description.abstractWe substantiate our preliminary description of the class I tryptophanyl-tRNA synthetase minimal catalytic domain with details of its construction, structure, and steady-state kinetic parameters. Generating that active fragment involved deleting 65% of the contemporary enzyme, including the anticodon-binding domain and connecting peptide 1, CP1, a 74-residue internal segment from within the Rossmann fold. We used protein design (Rosetta), rather than phylogenetic sequence alignments, to identify mutations to compensate for the severe loss of modularity, thus restoring stability, as evidenced by renaturation described previously and by 70-ns molecular dynamics simulations. Sufficient solubility to enable biochemical studies was achieved by expressing the redesigned Urzyme as a maltose-binding protein fusion. Michaelis-Menten kinetic parameters from amino acid activation assays showed that, compared with the native full-length enzyme, TrpRS Urzyme binds ATP with similar affinity. This suggests that neither of the two deleted structural modules has a strong influence on ground-state ATP binding. However, tryptophan has 103 lower affinity, and the Urzyme has comparably reduced specificity relative to the related amino acid, tyrosine. Molecular dynamics simulations revealed how CP1 may contribute significantly to cognate amino acid specificity. As class Ia editing domains are nested within the CP1, this finding suggests that this module enhanced amino acid specificity continuously, throughout their evolution. We call this type of reconstructed protein catalyst an Urzyme (Ur prefix indicates original, primitive, or earliest). It establishes a model for recapitulating very early steps in molecular evolution in which fitness may have been enhanced by accumulating entire modules, rather than by discrete amino acid sequence changes. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.en_HK
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_HK
dc.relation.ispartofJournal of Biological Chemistryen_HK
dc.rightsJournal of Biological Chemistry. Copyright © American Society for Biochemistry and Molecular Biology, Inc.-
dc.subject.meshComputer Simulationen_US
dc.subject.meshEvolution, Molecularen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshProtein Bindingen_US
dc.subject.meshTryptophan-tRNA Ligase - genetics - metabolismen_US
dc.titleTryptophanyl-tRNA synthetase urzyme: A model to recapitulate molecular evolution and investigate intramolecular complementationen_HK
dc.typeArticleen_HK
dc.identifier.emailHu, H:haohu@hku.hken_HK
dc.identifier.authorityHu, H=rp00707en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1074/jbc.M110.136911en_HK
dc.identifier.pmid20864539-
dc.identifier.pmcidPMC2992291en_US
dc.identifier.scopuseid_2-s2.0-78649653834en_HK
dc.identifier.hkuros184580en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-78649653834&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume285en_HK
dc.identifier.issue49en_HK
dc.identifier.spage38590en_HK
dc.identifier.epage38601en_HK
dc.identifier.eissn1083-351X-
dc.identifier.isiWOS:000284625600084-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridPham, Y=6603350759en_HK
dc.identifier.scopusauthoridKuhlman, B=6701347944en_HK
dc.identifier.scopusauthoridButterfoss, GL=6506454968en_HK
dc.identifier.scopusauthoridHu, H=7404097564en_HK
dc.identifier.scopusauthoridWeinreb, V=8735389700en_HK
dc.identifier.scopusauthoridCarter Jr, CW=7403317501en_HK

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