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

Hsueh-Wei Tseng; Tobias Baumann; Huan Sun; Yane-Shih Wang; Zoya Ignatova; Nediljko Budisa

doi:10.3390/molecules25194418

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

Molecules ◽

10.3390/molecules25194418 ◽

2020 ◽

Vol 25 (19) ◽

pp. 4418

Author(s):

Hsueh-Wei Tseng ◽

Tobias Baumann ◽

Huan Sun ◽

Yane-Shih Wang ◽

Zoya Ignatova ◽

...

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Binding Pocket ◽

Trna Synthetase ◽

Rational Approach ◽

Translation Efficiency ◽

Amino Acid Residues ◽

Trna Synthetases ◽

Methanosarcina Mazei ◽

Genetic Code Expansion

In 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 translation

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Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination

Biochemical Journal ◽

10.1042/bj20051249 ◽

2006 ◽

Vol 394 (2) ◽

pp. 399-407 ◽

Cited By ~ 20

Author(s):

Yunqing Liu ◽

Jing Liao ◽

Bin Zhu ◽

En-Duo Wang ◽

Jianping Ding

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Crystal Structures ◽

Active Site ◽

Binding Pocket ◽

Trna Synthetase ◽

Vital Role ◽

Common Mechanism ◽

Trna Synthetases ◽

Editing Domain

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels

Nucleic Acids Research ◽

10.1093/nar/gkaa055 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3071-3088

Author(s):

Matthew R McFarland ◽

Corina D Keller ◽

Brandon M Childers ◽

Stephen A Adeniyi ◽

Holly Corrigall ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurological Disorders ◽

Trna Synthetase ◽

Northern Blotting ◽

Amino Acid Starvation ◽

Starvation Response ◽

Kinase Activation ◽

Trna Synthetases ◽

Starvation Conditions

Abstract During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

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The Role of Orthogonality in Genetic Code Expansion

Life ◽

10.3390/life9030058 ◽

2019 ◽

Vol 9 (3) ◽

pp. 58 ◽

Cited By ~ 6

Author(s):

Pol Arranz-Gibert ◽

Jaymin R. Patel ◽

Farren J. Isaacs

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Genetic Code ◽

Cross Reactivity ◽

Elongation Factors ◽

Translational Machinery ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Genetic Code Expansion

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

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Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

Biological Chemistry ◽

10.1515/bc.2009.027 ◽

2009 ◽

Vol 390 (3) ◽

Cited By ~ 5

Author(s):

Takayuki K. Nemoto ◽

Toshio Ono ◽

Yu Shimoyama ◽

Shigenobu Kimura ◽

Yuko Ohara-Nemoto

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protease Activity ◽

Binding Pocket ◽

Catalytic Triad ◽

Amino Acid Residues ◽

Proteolytic Activities ◽

Hydrophobic Amino Acids ◽

The Difference

Abstract Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.

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Computational-Designed Enzyme for β-Tyrosine Production in Lignin Valorization

Catalysts ◽

10.3390/catal11111310 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1310

Author(s):

Fei Peng ◽

Habibu Aliyu ◽

André Delavault ◽

Ulrike Engel ◽

Jens Rudat

Keyword(s):

Amino Acids ◽

Value Chain ◽

Aromatic Amino Acids ◽

Catalytic Efficiency ◽

Building Blocks ◽

Binding Pocket ◽

Synthesis Process ◽

Taxus Chinensis ◽

Lignin Valorization ◽

Computational Enzyme Design

Lignin is an underutilized sustainable source of aromatic compounds. To valorize the low-value lignin monomers, we proposed an efficient strategy, involving enzymatic conversion from trans-p-hydroxycinnamic acids to generate valued-added canonical and non-canonical aromatic amino acids. Among them, β-amino acids are recognized as building blocks for bioactive natural products and pharmaceutical ingredients due to their attractive antitumor properties. Using computational enzyme design, the (R)-β-selective phenylalanine aminomutase from Taxus chinensis (TchPAM) was successfully mutated to accept β-tyrosine as the substrate, as well as to generate the (R)-β-tyrosine with excellent enantiopurity (ee > 99%) as the unique product from trans-p-hydroxycinnamic acid. Moreover, the kinetic parameters were determined for the reaction of four Y424 enzyme variants with the synthesis of different phenylalanine and tyrosine enantiomers. In the ammonia elimination reaction of (R)-β-tyrosine, the variants Y424N and Y424C displayed a two-fold increased catalytic efficiency of the wild type. In this work, a binding pocket in the active site, including Y424, K427, I431, and E455, was examined for its influence on the β-enantioselectivity of this enzyme family. Combining the upstream lignin depolymerization and downstream production, a sustainable value chain based on lignin is enabled. In summary, we report a β-tyrosine synthesis process from a monolignol component, offering a new way for lignin valorization by biocatalyst modification.

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High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

10.1101/2021.07.13.452272 ◽

2021 ◽

Author(s):

Jessica T. Stieglitz ◽

James A. Van Deventer

Keyword(s):

Genetic Code ◽

High Throughput ◽

Trna Synthetase ◽

Noncanonical Amino Acids ◽

Trna Synthetases ◽

E Coli ◽

Biological Studies ◽

Screening Strategies ◽

Genetic Code Expansion ◽

Translation Systems

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting (FACS) to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific/polyspecific properties are invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

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Quality control in tRNA charging -- editing of homocysteine.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2259 ◽

2011 ◽

Vol 58 (2) ◽

Cited By ~ 34

Author(s):

Hieronim Jakubowski

Keyword(s):

Amino Acids ◽

Quality Control ◽

Control Point ◽

Building Blocks ◽

Trna Synthetase ◽

Nutritional Deficiencies ◽

Homocysteine Thiolactone ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Living Organisms

All living organisms conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from a gene to protein product. One crucial 'quality control' point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. When differences in binding energies of amino acids to an aminoacyl-tRNA synthetase are inadequate, editing is used as a major determinant of enzyme selectivity. Some incorrect amino acids are edited at the active site before the transfer to tRNA (pre-transfer editing), while others are edited after transfer to tRNA at a separate editing site (post-transfer editing). Access of natural non-protein amino acids, such as homocysteine, homoserine, or ornithine to the genetic code is prevented by the editing function of aminoacyl-tRNA synthetases. Disabling editing function leads to tRNA mischarging errors and incorporation of incorrect amino acids into protein, which is detrimental to cell homeostasis and inhibits growth. Continuous homocysteine editing by methionyl-tRNA synthetase, resulting in the synthesis of homocysteine thiolactone, is part of the process of tRNA aminoacylation in living organisms, from bacteria to man. Excessive homocysteine thiolactone synthesis in hyperhomocysteinemia caused by genetic or nutritional deficiencies is linked to human vascular and neurological diseases.

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tRNA Synthetases Are Recruited to Yeast Ribosomes by rRNA Expansion Segment 7L but Do Not Require Association for Functionality

Non-Coding RNA ◽

10.3390/ncrna7040073 ◽

2021 ◽

Vol 7 (4) ◽

pp. 73

Author(s):

Nina Krauer ◽

Robert Rauscher ◽

Norbert Polacek

Keyword(s):

Ribosome Biogenesis ◽

Protein Biosynthesis ◽

Ribosomal Subunit ◽

Trna Synthetase ◽

Translation Efficiency ◽

Functional Roles ◽

Trna Synthetases ◽

Yeast Ribosomes ◽

Ribosome Association

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions.

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Antibacterial Activity of the Root Extracts of Garcinia Kola Against MDR Staphylococcus Aureus : Invitro and Insilico Studies

10.26434/chemrxiv.13649606.v1 ◽

2021 ◽

Author(s):

Abdulbasit Haliru Yakubu ◽

Muhammad Mustapha Muhammad

Keyword(s):

Staphylococcus Aureus ◽

Binding Pocket ◽

Docking Studies ◽

Trna Synthetase ◽

Root Extract ◽

Antibacterial Drug ◽

Economic Implication ◽

Amino Acid Residues ◽

Garcinia Kola ◽

Staph Aureus

MDR Staphylococcus aureus is an important bacteria with clinical and economic implication. Plants including Garcinia kola provides bioactive principles with diverse structural and biological features.. The n-Butanol fraction of G.kola root extract recorded the highest activity against MDR staph aureus (18.50±0.41) compared to the chloroform (10.00±2.12) and methanol (8.166±0.62) extarct, with no activity recorded with the n-Hexane extract. Analysis of this fraction on GC-MS recorded 14 phytoconstituents with varying structural composition; containing important scaffolds & motifs of benzoquinone, <a href="https://pubchem.ncbi.nlm.nih.gov/compound/imidazo[1,2-a]pyridine">imidazo[1,2-a]pyridine</a>, Chlorocarbazole and azetidine that present key pharmaceuticals as antibiotic and for drug development. Further inslico molecular docking studies of these compounds on antibacterial drug target; Tyrosyl-tRNA synthetase (PDB 1JIJ) from MDR staph aureus was documented. 9 compounds (CID_619544, CID_619583, CID_5732, CID_616643, CID_622021, CID_ 616496, CID_590350, CID_16486 and CID_66747) had good binding scores ranging from -4.63 to -7.08 kcal/mol; with CID_590350 having the highest score. The compounds formed various bonding with the 1JIJ amino acid residues including H-bond, van der waal and π interactions. CID_16486 and CID_66747 bind to the most active binding pocket (Drug score: 0.82 &0.72) while CID_619583 tend to bind outside the active binding pocket. They also have good pharmacokinetic and toxicity profile. Therefore, these compounds are considered as suitable prospective antibiotics against MDR Staphylococcus aureus after successful invitro and insilico experimental validation.

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