Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

Acevedo‐Rocha, Carlos G.; Hoesl, Michael Georg; Nehring, Sebastian; Royter, Marina; Wolschner, Christina; Wiltschi, Birgit; Antranikian, Garabed; Budiša, Nediljko

doi:10.1039/c3cy20712a

Cited by 43 publications

(29 citation statements)

References 16 publications

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“…Their incorporation in the Met-rich active site of Calmodulin lowered its activity, [42] whereas incorporation of Nle into Lipase from Thermoanaerobacter thermohydrosulfuricus (TTL) led to an enzyme highly active in the aqueous phase without the need for thermal activation [43] and enhanced stability against denaturing agents. [44] Other examples of Met analogue applications include the use of methoxinine to study the effects of Met oxidation on prion proteins, [45] of photo-methionine in protein-protein interaction studies, [46] and of homoallylglycine (Hag) for potential use in olefin metathesis. [47] Even Met analogues which are generally translationally inactive can be incorporated by simple overexpression of MetRS, as Tirrell and coworkers showed with trans-crotylglycine (Tgc), cis-crotylglycine (Ctg), 2-aminoheptanoic acid, norvaline, 2-butynylglycine, allylglycine [48][49][50] and S-allylhomocysteine.…”

Section: Global Reassignment Of the Aug Codonmentioning

confidence: 99%

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNA fMet initiates protein synthesis (starting AUG codon), whereas elongator tRNA Met participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNA Met pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNA Met from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNA Pyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

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Section: Global Reassignment Of the Aug Codonmentioning

confidence: 99%

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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“…The list of expected tryptic digests of mDHFR was obtained using the online server ExPASy SIB Bioinformatics Resource Portal. The peptide fragment "NGDLPWPPLRNEFK" (residues [19][20][21][22][23][24][25][26][27][28][29][30][31][32], containing the Phe residue at position 31 served as the reference. The expected monoisotopic mass of the reporter peptide is 1682.9 (m/z).…”

Section: Site-specific Incorporation Of 2nal At Position 31 Of Mdhfr mentioning

confidence: 99%

“…Several techniques using non-natural amino acids have developed [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Residue-specific incorporation of non-natural amino acids is recently demonstrated as a very useful tool for redesign of industrial enzymes [27], or bio-catalysts in general [28], and even useful to perform enzyme reactions in hostile environments [29]. Alternatively, site-specific incorporation in vivo techniques including stop codon suppression in vivo (SCS) shows great potential for enzyme engineering.…”

Section: Introductionmentioning

confidence: 98%

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

Zheng

Lim

Kwon

2015

Biochemical Engineering Journal

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“…Die verbleibende hydrolytische Aktivität der von Acevedo‐Rocha et al. durch SPI erzeugten TTL‐Varianten wurde unter Verwendung verschiedener organischer Lösungsmittel, Metallionen, grenzflächenaktiver Substanzen, Inhibitoren, reduzierender Bedingungen, alkylierender und denaturierender Reagenzien studiert. Zu den bemerkenswertesten Auswirkungen gehörte, dass TTL eine partielle Aktivität nach Behandlung mit 90 % Pyridin behielt, wenn es global mit den Trp‐Analoga 4‐Amino‐, 4‐Fluor‐ oder 7‐Aza‐Trp (4AmW, 4FW und 7AW, Abbildung ) oder mit dem Met‐Analogon Aha modifiziert wurde.…”

Section: Neue Chemische Funktionalitäten: Was Haben Wir Gelernt?unclassified

Biokatalyse mit nicht‐natürlichen Aminosäuren: Enzymologie trifft Xenobiologie

et al. 2017

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Das Ziel der Xenobiologie ist das Design von biologischen Systemen, die mit nicht‐natürlichen biochemischen Funktionen ausgestattet sind, während die Enzymologie sich mit dem Verständnis von Enzymen auseinandersetzt, den Routinewerkzeugen der Biokatalyse. In der Biokatalyse werden Enzyme und Organismen dazu verwendet, um nützliche und leistungsfähige Biotransformationen in der synthetischen Chemie und Biotechnologie durchzuführen. In den vergangenen Jahren sind die Auswirkungen des Einbaus von nicht‐kanonischen Aminosäuren (nkAAs) in Enzyme, welche potentiell interessante Anwendung für die Biokatalyse aufweisen, mit gesteigerter Aufmerksamkeit untersucht worden. Mit diesem Aufsatz wollen wir einen Überblick über die Effekte und Auswirkungen neuer chemischer Funktionalitäten geben, mit denen Proteine ausgestattet wurden, um eine Vielzahl von Aspekten der enzymatischen Biokatalyse zu verbessern. Ein weiteres Hauptaugenmerk liegt auf zukünftigen Forschungsperspektiven, welche die nicht‐natürliche Mutagenese durch die Möglichkeiten des herkömmlichen Protein‐Engineerings ergänzen werden, um neue und vielseitige Biokatalysatoren mit Anwendungen in der synthetischen organischen Chemie und Biotechnologie zu generieren.

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Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

Cited by 43 publications

References 16 publications

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

Biokatalyse mit nicht‐natürlichen Aminosäuren: Enzymologie trifft Xenobiologie

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