Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

dc.contributor.authorTseng, Hsueh-Wei
dc.contributor.authorBaumann, Tobias
dc.contributor.authorSun, Huan
dc.contributor.authorWang, Yane-Shih
dc.contributor.authorIgnatova, Zoya
dc.contributor.authorBudisa, Nediljko
dc.date.accessioned2020-11-04T15:29:45Z
dc.date.available2020-11-04T15:29:45Z
dc.date.issued2020-09-25
dc.date.updated2020-10-08T09:22:59Z
dc.description.abstractIn protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translationen
dc.description.sponsorshipTU Berlin, Open-Access-Mittel – 2020en
dc.identifier.eissn1420-3049
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/11832
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-10722
dc.language.isoenen
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en
dc.subject.ddc540 Chemie und zugeordnete Wissenschaftende
dc.subject.otheraromatic amino acid analogsen
dc.subject.othersite-specific incorporationen
dc.subject.othergenetic code expansionen
dc.subject.otherribosomal translationen
dc.subject.othernon-canonical amino acidsen
dc.subject.otherncAAsen
dc.subject.otherorthogonal pairsen
dc.subject.otherMethanosarcina mazei pyrrolysyl-tRNA synthetaseen
dc.subject.otherMmPylRSen
dc.titleExpanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acidsen
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.articlenumber4418en
dcterms.bibliographicCitation.doi10.3390/molecules25194418en
dcterms.bibliographicCitation.issue19en
dcterms.bibliographicCitation.journaltitleMoleculesen
dcterms.bibliographicCitation.originalpublishernameMDPIen
dcterms.bibliographicCitation.originalpublisherplaceBaselen
dcterms.bibliographicCitation.volume25en
tub.accessrights.dnbfreeen
tub.affiliationFak. 2 Mathematik und Naturwissenschaften::Inst. Chemie::FG Biokatalysede
tub.affiliation.facultyFak. 2 Mathematik und Naturwissenschaftende
tub.affiliation.groupFG Biokatalysede
tub.affiliation.instituteInst. Chemiede
tub.publisher.universityorinstitutionTechnische Universität Berlinen

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