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The FEBS Journal Jun 2023Alanine racemases (ALRs) are essential for d-alanine (d-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site....
Alanine racemases (ALRs) are essential for d-alanine (d-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short-chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in K , indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylate-binding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate-binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K-mediated activation in the presence of d-Ala at high concentrations, implying that d-Ala also exhibits low-affinity binding in the first carboxylate-binding site and prevents carboxylate/G134K-induced activation. Such regulation of enzyme activity by carboxylates and d-Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.
Topics: Alanine Racemase; Alanine; Lysine; Binding Sites; Catalytic Domain; Carboxylic Acids; Kinetics
PubMed: 36732053
DOI: 10.1111/febs.16745 -
Journal of Comparative Effectiveness... Dec 2020The race to find an effective treatment for coronavirus disease 2019 (COVID-19) is still on, with only two treatment options currently authorized for emergency use... (Review)
Review
The race to find an effective treatment for coronavirus disease 2019 (COVID-19) is still on, with only two treatment options currently authorized for emergency use and/or recommended for patients hospitalized with severe respiratory symptoms: low-dose dexamethasone and remdesivir. The USA decision to stockpile the latter has resulted in widespread condemnation and in similar action being taken by some other countries. In this commentary we discuss whether stockpiling remdesivir is justified in light of the currently available evidence.
Topics: Adenosine Monophosphate; Alanine; Antiviral Agents; Humans; International Cooperation; Internationality; SARS-CoV-2; Strategic Stockpile; United States; COVID-19 Drug Treatment
PubMed: 33274643
DOI: 10.2217/cer-2020-0174 -
Analytical Chemistry Sep 2020Tissue pH is tightly regulated in vivo, being a sensitive physiological biomarker. Advent of dissolution dynamic nuclear polarization (DNP) and its translation to humans...
Tissue pH is tightly regulated in vivo, being a sensitive physiological biomarker. Advent of dissolution dynamic nuclear polarization (DNP) and its translation to humans stimulated development of pH-sensitive agents. However, requirements of DNP probes such as biocompatibility, signal sensitivity, and spin-lattice relaxation time (T) complicate in vivo translation of the agents. Here, we developed a C-labeled alanine derivative, [1-C]-l-alanine ethyl ester, as a viable DNP probe whose chemical shift is sensitive to the physiological pH range, and demonstrated the feasibility in phantoms and rat livers in vivo. Alanine ethyl ester readily crosses cell membrane while simultaneously assessing extracellular and intracellular pH in vivo. Following cell transport, [1-C]-l-alanine ethyl ester is instantaneously hydrolyzed to [1-C]-l-alanine, and subsequently metabolized to [1-C]lactate and [C]bicarbonate. The pH-insensitive alanine resonance was used as a reference.
Topics: Alanine; Animals; Esters; Hydrogen-Ion Concentration; Male; Models, Animal; Rats; Rats, Wistar
PubMed: 32786486
DOI: 10.1021/acs.analchem.0c01568 -
Science (New York, N.Y.) Mar 2024Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In...
Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.
Topics: Animals; Administration, Oral; Ebolavirus; Hemorrhagic Fever, Ebola; Macaca fascicularis; Nucleosides; Adenosine Monophosphate; Alanine; Prodrugs; Antiviral Agents
PubMed: 38484056
DOI: 10.1126/science.adk6176 -
Origins of Life and Evolution of the... Dec 2021After pre concentration of monomers, polymerization is the second most important step for molecular evolution. The formation of peptides is an important issue for...
After pre concentration of monomers, polymerization is the second most important step for molecular evolution. The formation of peptides is an important issue for prebiotic chemistry and consequently for the origin of life. In this work, goethite was synthesized by two different routes, named goethite-I and goethite-II. Although both samples are goethite, Far-FT-IR spectroscopy and EPR spectroscopy showed differences between them, and these differences had an effect on the polymerization of glycine and alanine. For the amino acid polymerization, three protocols were used, that resembled prebiotic Earth conditions: a) amino acid plus goethite were mixed and heated at 90 °C for 10 days in solid state, b) a wet impregnation of the amino acid in the goethite, with subsequent heating at 90 °C for 10 days in solid state, and c) 10 wet/dry cycles each one for 24 h at 90 °C. Experiments with glycine plus goethite-II, using protocols B and C, produced only Gly-Gly. In addition, for the C protocol the amount of Gly-Gly synthesized was 3 times higher than the amount of Ala-Ala. Goethite-I presented a decrease in the EPR signal, when it was submitted to the protocols with and without amino acids. It is probable the decrease in the intensity of the EPR signal was due to a decrease in the imperfections of the mineral. For all protocols the mixture of alanine plus goethite-I or goethite-II produced c(Ala-Ala). However, for wet/dry cycles, protocol C presented higher yields (p < 0.05). In addition, Ala-Ala was produced using protocols A and C. The c(Ala-Ala) formation fitted a zero-order kinetic equation model. The surface areas of goethite-I and goethite-II were 35 m g and 37 m g, respectively. Thermal analysis indicated that the mineral changes the thermal behavior of the amino acids. The main reactions for the thermal decomposition of glycine were deamination and dehydration and for alanine was deamination.
Topics: Alanine; Glycine; Iron Compounds; Minerals; Polymerization; Spectroscopy, Fourier Transform Infrared
PubMed: 35064872
DOI: 10.1007/s11084-021-09618-z -
Journal of Computational Chemistry Jan 2022We determine the proton affinity (PA) and gas-phase basicity (GB) of amino acid α-alanine at a chemically accurate level by performing explicitly-correlated...
We determine the proton affinity (PA) and gas-phase basicity (GB) of amino acid α-alanine at a chemically accurate level by performing explicitly-correlated CCSD(T)-F12b/aug-cc-pVDZ geometry optimizations and normal mode vibrational frequency calculations as well as CCSD(T)-F12b/aug-cc-pVTZ energy computations at the possible neutral and protonated geometries. Temperature effects at 298.15 K considering translational, rotational, and vibrational enthalpy and entropy corrections are obtained via standard statistical mechanics utilizing the molecular geometries and the harmonic vibrational energy levels. Both the amino nitrogen (N) and the carbonyl oxygen (O) atoms are proven to be potential protonation sites and a systematic conformational search reveals 3 N- and 9 O-protonated conformers in the 0.00-7.88 and 25.43-30.43 kcal/mol energy ranges at 0 K, respectively. The final computed PA and GB values at (0)298.15 K in case of N-protonation are (214.47)216.80 and 207.07 kcal/mol, respectively, whereas the corresponding values for O-protonation are (189.04)190.63 and 182.31 kcal/mol. The results of the benchmark high-level coupled-cluster computations are utilized to assess the accuracy of several lower-level cost-effective methods such as MP2 and density functional theory with various functionals (SOGGA11-X, M06-2X, PBE0, B3LYP, M06, TPSS).
Topics: Alanine; Nitrogen; Oxygen; Protons; Quantum Theory; Temperature
PubMed: 34676890
DOI: 10.1002/jcc.26767 -
International Journal of Molecular... Nov 2019A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA... (Review)
Review
A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elusive. Here we describe a model for this process by following the scheme previously proposed by Hartman and Smith, which suggests gradual expansion of the coding space as GC-GCA-GCAU genetic code. We point out a correlation of this scheme with the hierarchy of the protein folding. The model follows the sequence of steps in the process of the amino acid recruitment and fits well with the co-evolution and coenzyme handle theories. While the starting set (GC-phase) was responsible for the nucleotide biosynthesis processes, in the second phase alanine-based amino acids (GCA-phase) were recruited from the core metabolism, thereby providing a standard secondary structure, the α-helix. In the final phase (GCAU-phase), the amino acids were appended to the already existing architecture, enabling tertiary fold and membrane interactions. The whole scheme indicates strongly that the choice for the alanine core was done at the GCA-phase, while glycine and proline remained rudiments from the GC-phase. We suggest that the Protein World should rather be considered the Alanine World, as it predominantly relies on the alanine as the core chemical scaffold.
Topics: Alanine; Amino Acids; Animals; Evolution, Molecular; Genetic Code; Humans; Protein Biosynthesis; Protein Folding; Proteins
PubMed: 31694194
DOI: 10.3390/ijms20215507 -
Applied and Environmental Microbiology May 2022d-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. ()-transaminase [()-TA]-catalyzed asymmetric...
d-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. ()-transaminase [()-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of d-alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential ()-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using ()-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to d-alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of d-alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of d-alanine operating with high productivity. d-Alanine is an important compound with many valuable applications. Its asymmetric synthesis employing ()-ω-TA is considered an attractive choice. According to the stereoselectivity, ω-TAs have either ()- or ()-enantiopreference. There has been a variety of literature regarding screening, characterizing, and molecular modification of ()-ω-TAs; in contrast, the research about ()-ω-TA has lagged behind. In this work, we identify several ()-ω-TAs and succeeded in creating mutant F113T, which showed not only better efficiency toward pyruvate but also higher thermostability compared with the original enzyme. The obtained original enzymes and positive mutants displayed important application value for pushing symmetric synthesis of d-alanine to a higher level.
Topics: Alanine; Amino Acids; Ascomycota; Catalytic Domain; Pyruvic Acid; Transaminases
PubMed: 35465694
DOI: 10.1128/aem.00062-22 -
Infection Jun 2021COVID-19 is an infectious disease caused by a novel β-coronavirus, belonging to the same subgenus as the Severe Acute Respiratory Syndrome (SARS) virus. Remdesivir, an... (Review)
Review
COVID-19 is an infectious disease caused by a novel β-coronavirus, belonging to the same subgenus as the Severe Acute Respiratory Syndrome (SARS) virus. Remdesivir, an investigational broad-spectrum antiviral agent has previously demonstrated in vitro activity against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and in vivo efficacy against other related coronaviruses in animal models. Its safety profile has been tested in a compassionate use setting for patients with COVID-19. The current therapeutic studies demonstrate clinical effectiveness of remdesivir in COVID-19 patients by shortening time to clinical recovery, and hospital stay. In this review, we critically analyze the current evidence of remdesivir against COVID-19 and dissect the aspects over its safety and efficacy. Based on existing data, remdesivir can be regarded as a potential therapeutic agent against COVID-19. Further large-scale, randomized placebo-controlled clinical trials are, however, awaited to validate these findings.
Topics: Adenosine Monophosphate; Alanine; Animals; Antiviral Agents; COVID-19; Humans; SARS-CoV-2; Time Factors; Treatment Outcome; COVID-19 Drug Treatment
PubMed: 33389708
DOI: 10.1007/s15010-020-01557-7 -
Metallomics : Integrated Biometal... Dec 2020Low molecular weight selenium containing metabolites in the leaves of the selenium hyperaccumulator Cardamine violifolia (261 mg total Se per kg d.w.) were targeted in...
Low molecular weight selenium containing metabolites in the leaves of the selenium hyperaccumulator Cardamine violifolia (261 mg total Se per kg d.w.) were targeted in this study. One dimensional cation exchange chromatography coupled to ICP-MS was used for purification and fractionation purposes prior to LC-Unispray-QTOF-MS analysis. The search for selenium species in full scan spectra was assisted with an automated mass defect based filtering approach. Besides selenocystathionine, selenohomocystine and its polyselenide derivative, a total number of 35 water soluble selenium metabolites other than selenolanthionine were encountered, including 30 previously unreported compounds. High occurrence of selenium containing hexoses was observed, together with the first assignment of N-glycoside derivatives of selenolanthionine. Quantification of the most abundant selenium species, selenolanthionine, was carried out with an ion pairing LC - post column isotope dilution ICP-MS setup, which revealed that this selenoamino acid accounted for 30% of the total selenium content of the leaf (78 mg (as Se) per kg d.w.).
Topics: Alanine; Cardamine; Cystathionine; Homocystine; Organoselenium Compounds; Plant Leaves; Selenium; Solubility; Water
PubMed: 33165451
DOI: 10.1039/d0mt00216j