An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids
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ABSTRACT:
SUBMITTER: Liu K
PROVIDER: S-EPMC10991234 | biostudies-literature | 2023 Dec
REPOSITORIES: biostudies-literature
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Project description:By evolving the N-terminal domain of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) that directly interacts with tRNAPyl , a mutant clone displaying improved amber-suppression efficiency for the genetic incorporation of Nϵ -(tert-butoxycarbonyl)-l-lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of Nϵ -acetyl-l-lysine and Nϵ -(4-azidobenzoxycarbonyl)-l-δ,ϵ-dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N-terminal domain of PylRS that was separated from its catalytic domain for charging tRNAPyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system-based noncanonical amino-acid mutagenesis.
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Project description:A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUA(Pyl), a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.
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Project description:Post-translational modifications regulate protein structure and function. Lysine benzoylation is a newly discovered histone modification with unique physiological relevance. To construct proteins with this modification site-specifically, we generated orthogonal tRNAPyl -MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl-tRNA synthetase, allowing the genetic incorporation of ϵ-N-benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation.
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Project description:We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs from Methanomassiliicocales that are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivo activity in the Methanosarcina mazei ( Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/PyltRNA pairs, or their evolved variants [including Methanomethylophilus alvus ( Ma) PylRS/ MaPyltRNA(6)CUA], are mutually orthogonal to the MmPylRS/ MmPyltRNA pair, and the active sites of the Mm pair and Ma pair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/ MaPyltRNA(6)CUA is orthogonal to the aaRSs and tRNAs in mammalian cells and directs efficient incorporation of ncAAs into proteins. Moreover, we confirm that the MaPylRS/ MaPyltRNA(6) and MmPylRS/ MmPyltRNA pairs are mutually orthogonal in mammalian cells and demonstrates that these pairs can be used to encode distinct ncAAs into a protein in mammalian cells. Thus, the MaPylRS/ MaPyltRNA(6)CUA pair provides an additional pair that is orthogonal in both E. coli and mammalian systems and is mutually orthogonal to the most widely used system for genetic code expansion. Our results provide a foundation for expanding the scope of genetic code expansion and may also facilitate strategies for proteome-wide ncAA tagging with mutually orthogonal systems.
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Project description:Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.
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Project description:Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmPyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmPyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/PyltRNACUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/PyltRNA pairs that are mutually orthogonal to the MmPylRS/MmPyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.
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Project description:We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.
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Project description:Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme's anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.
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Project description:When coexpressed with its cognate amber suppressing tRNACUAPyl(CUA), a pyrrolysyltRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro,ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone,alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.
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Project description:The pyrrolysyl-tRNA synthetase (PylRS) is widely used to incorporate noncanonical amino acids (ncAAs) into proteins. However, most of ncAA-containing protein yields remain low due to the limited activity of PylRS variants. Here, we apply machine learning (ML) to engineer the tRNA-binding domain of PylRS. The FFT-PLSR model is first applied to explore pairwise combinations of 12 single mutations, generating a variant Com1-IFRS with an 11-fold increase in stop codon suppression efficiency. Deep learning models ESM-1v, Mutcompute, and ProRefiner then identify new mutation sites. Applying FFT-PLSR on these sites yields a variant Com2-IFRS showing a 30.8-fold increase in stop codon suppression efficiency. Transplanting these mutations into 7 other PylRS-derived synthetases improved ncAA-containing protein yield by up to 1149.7-fold. Molecular dynamics simulations are used to explore the molecular change caused by the mutations. This paper presents improved PylRS variants and a machine learning framework for optimizing the enzyme activity.
2025-07-25 | PXD065336 | Pride