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RNA Biology Jan 2023Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA...
Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNA and the anticodon plays no role in charging, tRNA variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNA anticodon variants on the growth of . Overall, 36 tRNA anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNA variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNA anticodon variants.
Topics: Anticodon; RNA, Transfer, Ala; RNA, Transfer; Codon; Alanine; Protein Biosynthesis
PubMed: 37776539
DOI: 10.1080/15476286.2023.2257471 -
Indian Journal of Pediatrics Aug 2022
Topics: Adenosine Monophosphate; Alanine; Chemical and Drug Induced Liver Injury, Chronic; Humans; Infant, Newborn; COVID-19 Drug Treatment
PubMed: 35583631
DOI: 10.1007/s12098-022-04237-6 -
Chemical & Pharmaceutical Bulletin 2019Rebamipide is a therapeutic agent for gastric ulcers and chronic gastritis. Hypobromous acid (HOBr) is generated not only by eosinophils but also by neutrophils in the...
Rebamipide is a therapeutic agent for gastric ulcers and chronic gastritis. Hypobromous acid (HOBr) is generated not only by eosinophils but also by neutrophils in the presence of bromide ions in the plasma. At inflammation sites, rebamipide may encounter and react with HOBr to generated various products. When rebamipide was incubated with reagent HOBr in potassium phosphate buffer at pH 4.7 and 37°C for 4 h, several products were generated. A major product was identified as 3-bromorebamipide, a novel compound. Rebamipide does not react with hypochlorous acid (HOCl). However, when rebamipide was incubated with HOCl in the presence of NaBr, 3-bromorebamipide was generated in a dose-dependent manner, probably because of formation of HOBr. These results suggest that 3-bromorebamipide may generate from rebamipide at inflammation sites in humans.
Topics: Alanine; Bromates; Humans; Hypochlorous Acid; Molecular Structure; Quinolones
PubMed: 31582637
DOI: 10.1248/cpb.c19-00384 -
Biochemical Society Transactions Oct 2020The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as... (Review)
Review
The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as hydrogen bridges or is induced by ligand-receptor interactions. The conformational freedom of a peptide can be constrained by intramolecular cross-links. Conformational constraints enhance the receptor specificity, may lead to biased activity and confer proteolytic resistance to peptidic GPCR agonists. Chemical synthesis allows to introduce a variety of cross-links into a peptide and is suitable for bulk production of relatively simple lead peptides. Lanthionines are thioether bridged alanines of which the two alanines can be introduced at different distances in chosen positions in a peptide. Thioether bridges are much more stable than disulfide bridges. Biosynthesis of lanthionine-constrained peptides exploiting engineered Gram-positive or Gram-negative bacteria that contain lanthionine-introducing enzymes constitutes a convenient method for discovery of lanthionine-stabilized GPCR agonists. The presence of an N-terminal leader peptide enables dehydratases to dehydrate serines and threonines in the peptide of interest after which a cyclase can couple the formed dehydroamino acids to cysteines forming (methyl)lanthionines. The leader peptide also guides the export of the formed lanthionine-containing precursor peptide out of Gram-positive bacteria via a lanthipeptide transporter. An engineered cleavage site in the C-terminus of the leader peptide allows to cleave off the leader peptide yielding the modified peptide of interest. Lanthipeptide GPCR agonists are an emerging class of therapeutics of which a few examples have demonstrated high efficacy in animal models of a variety of diseases. One lanthipeptide GPCR agonist has successfully passed clinical Phase Ia.
Topics: Alanine; Animals; Anti-Bacterial Agents; Bacterial Proteins; Disulfides; Drug Discovery; GTP-Binding Proteins; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Lanthanoid Series Elements; Membrane Transport Proteins; Mice; Peptide Hydrolases; Peptides; Protein Domains; Protein Processing, Post-Translational; Protein Sorting Signals; Rats; Receptors, G-Protein-Coupled; Substrate Specificity; Sulfides
PubMed: 33125486
DOI: 10.1042/BST20200427 -
American Family Physician Feb 2022
Topics: Adenosine Monophosphate; Alanine; Antiviral Agents; Humans; Treatment Outcome; COVID-19 Drug Treatment
PubMed: 35166509
DOI: No ID Found -
Protein Science : a Publication of the... May 2022The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on...
The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein-protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein.
Topics: Alanine; Alleles; Mutation; Transaminases
PubMed: 35481644
DOI: 10.1002/pro.4303 -
Journal of Enzyme Inhibition and... Dec 2018O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5'-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is...
O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5'-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff's base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.
Topics: Alanine; Cysteine Synthase; Dose-Response Relationship, Drug; Enzyme Inhibitors; Molecular Structure; Salmonella enterica; Structure-Activity Relationship
PubMed: 30251899
DOI: 10.1080/14756366.2018.1504040 -
The Journal of Physical Chemistry. B Apr 2022Dipeptides, the prototype peptides, exist in both linear (-) and cyclo (-) structures. Since the first mass spectrometry experiments, it has been observed that some...
Dipeptides, the prototype peptides, exist in both linear (-) and cyclo (-) structures. Since the first mass spectrometry experiments, it has been observed that some -structures may turn into the cyclo ones, likely via a temperature-induced process. In this work, combining several different experimental techniques (mass spectrometry, infrared and Raman spectroscopy, and thermogravimetric analysis) with tight-binding and ab initio simulations, we provide evidence that, in the case of l-phenylalanyl-l-alanine, an irreversible cyclization mechanism, catalyzed by water and driven by temperature, occurs in the condensed phase. This process can be considered as a very efficient strategy to improve dipeptide stability by turning the comparatively fragile linear structure into the robust and more stable cyclic one. This mechanism may have played a role in prebiotic chemistry and can be further exploited in the preparation of nanomaterials and drugs.
Topics: Alanine; Cyclization; Dipeptides; Peptides; Phenylalanine
PubMed: 35438499
DOI: 10.1021/acs.jpcb.1c10736 -
Biomedicine & Pharmacotherapy =... Oct 2020
Review
Topics: Adenosine Monophosphate; Adult; Alanine; Antiviral Agents; Female; Humans; Pregnancy; COVID-19 Drug Treatment
PubMed: 32707440
DOI: 10.1016/j.biopha.2020.110532 -
Scientific Reports Aug 2019Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a...
Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.
Topics: Alanine; Alanine Dehydrogenase; Biocatalysis; Cell-Free System; Enzymes; Kinetics; NAD
PubMed: 31409820
DOI: 10.1038/s41598-019-48151-y