Nickel-Catalyzed Cross-Coupling of Amino-Acid-Derived Alkylzinc Reagents with Alkyl Bromides/Chlorides: Access to Diverse Unnatural Amino Acids

2021 ◽  
Author(s):  
Fei-Hu Gou ◽  
Ming-Jian Ma ◽  
An-Jun Wang ◽  
Liang Zhao ◽  
Haoyang Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Cross Coupling ◽  
Alkyl Bromides

scholarly journals 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 ◽  
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.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

scholarly journals High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

2013 ◽  
Vol 80 (5) ◽  
pp. 1718-1725 ◽  
Author(s):  
Masaomi Minaba ◽  
Yusuke Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Expression System ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Translational Efficiency ◽  
Site Specific ◽  
Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

scholarly journals Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

2007 ◽  
Vol 283 (3) ◽  
pp. 1525-1533 ◽  
Author(s):  
Shixin Ye ◽  
Caroline Köhrer ◽  
Thomas Huber ◽  
Manija Kazmi ◽  
Pallavi Sachdev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
G Protein Coupled Receptors ◽  
Calcium Flux ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids

2008 ◽  
Vol 50 (2-4) ◽  
pp. 93-98 ◽  
Author(s):  
Antonio Caligiuri ◽  
Paola D’Arrigo ◽  
Elena Rosini ◽  
Giuseppe Pedrocchi-Fantoni ◽  
Davide Tessaro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

scholarly journals Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

2019 ◽  
Author(s):  
Xinxin Zheng ◽  
Yinglu Cui ◽  
Tao Li ◽  
Ruifeng Li ◽  
Lu Guo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Catalytic Mechanism ◽  
Chiral Amines ◽  
Branched Chain Amino Acid ◽  
Chiral Amino Acids ◽  
Branched Chain ◽  
Substrate Spectrum

AbstractAminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

Incorporation of Unnatural Amino Acids into Cytochrome c3 and Specific Viologen Binding to the Unnatural Amino Acid

ChemBioChem ◽  
2006 ◽  
Vol 7 (12) ◽  
pp. 1853-1855 ◽  
Author(s):  
Shin Iida ◽  
Noriyuki Asakura ◽  
Kenji Tabata ◽  
Ichiro Okura ◽  
Toshiaki Kamachi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
Cytochrome C3

Synthesis of Unnatural Amino Acids via Suzuki Cross-Coupling of Enantiopure Vinyloxazolidine Derivatives

2000 ◽  
Vol 2 (8) ◽  
pp. 1089-1092 ◽  
Author(s):  
Mark Sabat ◽  
Carl R. Johnson
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Cross Coupling

Enzymatic Conversion of Unnatural Amino Acids by YeastD-Amino Acid Oxidase

2006 ◽  
Vol 348 (15) ◽  
pp. 2183-2190 ◽  
Author(s):  
Antonio Caligiuri ◽  
Paola D'Arrigo ◽  
Elena Rosini ◽  
Davide Tessaro ◽  
Gianluca Molla ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Acid Oxidase ◽  
Enzymatic Conversion ◽  
Amino Acid Oxidase

Synthetic studies of novel ninhydrin analogs

2001 ◽  
Vol 79 (11) ◽  
pp. 1632-1654 ◽  
Author(s):  
Richard R Hark ◽  
Diane B Hauze ◽  
Olga Petrovskaia ◽  
Madeleine M Joullié
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cross Coupling ◽  
Coupling Reactions ◽  
Latent Fingerprints ◽  
Cross Coupling Reactions ◽  
Essential Tool ◽  
Porous Surfaces ◽  
Synthetic Studies ◽  
Sulfur Containing

Ninhydrin is an essential tool in the analysis of amino acids, peptides, and proteins, and the preferred reagent for the detection of latent fingerprints on porous surfaces. The goal of this investigation was to prepare ninhydrin analogs with enhanced chromogenic and fluorogenic properties. Target compounds included structures with extended conjugation and (or) with the presence of sulfur-containing moieties. We have devised general convergent routes for novel heterocyclic and aryl-substituted ninhydrin analogs for use as reagents for amino acid detection.Key words: ninhydrin analogs, synthesis, ketals, Suzuki cross-coupling reactions, Stille cross-coupling reactions.

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