Alanine Formation Research Articles

Persulfide Transfer to SufE Activates the Half-Sites Reactivity of the E. coli Cysteine Desulfurase SufS.

The Escherichia coli cysteine desulfurase SufS (EcSufS) is a dimeric, PLP-dependent enzyme responsible for sulfur mobilization in the SUF Fe-S cluster bioassembly pathway. The enzyme uses cysteine as a sulfur source and generates alanine and a covalent persulfide located on an active site of cysteine. Optimal in vitro activity of EcSufS requires the presence of the transpersulfurase protein, EcSufE, and a strong reductant. Here, presteady-state and single-turnover kinetics are used to investigate the mechanism of EcSufS activation by EcSufE. In the absence of EcSufE, EcSufS exhibits a presteady-state burst of product production with an amplitude of ∼0.4 active site equivalents, consistent with a half-sites reactivity. KinTek Explorer was used to isolate the first turnover of alanine formation and fit the data with a simplified kinetic mechanism with steps for alanine formation (k3) and a net rate constant for the downstream steps (k5). Using this treatment, microscopic rate constants of 2.3 ± 0.5 s-1 and 0.10 ± 0.01 s-1 were determined for k3 and k5, respectively. The inclusion of EcSufE in the reaction results in a similar rate constant for k3 but induces a 10-fold enhancement of k5 to 1.1 ± 0.2 s-1, such that both steps are partially rate-determining. The most likely downstream step where EcSufE could exert influence on EcSufS activity is the removal of the persulfide intermediate. Importantly, this step appears to serve as a limiting feature in the half-sites activity such that activating persulfide transfer allows for rapid shifting between active sites. Single-turnover assays show that the presence of EcSufE slightly slowed the rates of alanine-forming steps, suggesting it does not activate steps in the desulfurase half reaction.

Atomic Ruthenium‐Promoted Cadmium Sulfide for Photocatalytic Production of Amino Acids from Biomass Derivatives

AbstractAmino acids are the building blocks of proteins and are widely used as important ingredients for other nitrogen‐containing molecules. Here, we report the sustainable production of amino acids from biomass‐derived hydroxy acids with high activity under visible‐light irradiation and mild conditions, using atomic ruthenium‐promoted cadmium sulfide (Ru1/CdS). On a metal basis, the optimized Ru1/CdS exhibits a maximal alanine formation rate of 26.0 molAla ⋅ gRu−1 ⋅ h−1, which is 1.7 times and more than two orders of magnitude higher than that of its nanoparticle counterpart and the conventional thermocatalytic process, respectively. Integrated spectroscopic analysis and density functional theory calculations attribute the high performance of Ru1/CdS to the facilitated charge separation and O−H bond dissociation of the α‐hydroxy group, here of lactic acid. The operando nuclear magnetic resonance further infers a unique “double activation” mechanism of both the CH−OH and CH3−CH−OH structures in lactic acid, which significantly accelerates its photocatalytic amination toward alanine.

Atomic Ruthenium-Promoted Cadmium Sulfide for Photocatalytic Production of Amino Acids from Biomass Derivatives.

Amino acids are the building blocks of proteins and are widely used as important ingredients for other nitrogen-containing molecules. Here, we report the sustainable production of amino acids from biomass-derived hydroxy acids with high activity under visible-light irradiation and mild conditions, using atomic ruthenium-promoted cadmium sulfide (Ru1/CdS). On a metal basis, the optimized Ru1/CdS exhibits a maximal alanine formation rate of 26.0 molAla ⋅ gRu -1 ⋅ h-1, which is 1.7 times and more than two orders of magnitude higher than that of its nanoparticle counterpart and the conventional thermocatalytic process, respectively. Integrated spectroscopic analysis and density functional theory calculations attribute the high performance of Ru1/CdS to the facilitated charge separation and O-H bond dissociation of the α-hydroxy group, here of lactic acid. The operando nuclear magnetic resonance further infers a unique "double activation" mechanism of both the CH-OH and CH3-CH-OH structures in lactic acid, which significantly accelerates its photocatalytic amination toward alanine.

Alanine Formation in a Zero-Gap Flow Cell and the Role of TiO2/Ti Electrocatalysts.

The electrochemical synthesis of -amino acids at room temperature and pressure is a sustainable alternative to conventional methods like microbial fermentation and Strecker synthesis. A custom-built zero-gap flow electrolyzer was used to study the electrosynthesis of alanine via the electrocatalytic reductive amination (ERA) of the corresponding biomass-derivable -keto acid precursor - pyruvic acid (PA), and hydroxylamine (NH2OH) at very low pH. Non-toxic, abundant, and easy to prepare TiO2/Ti electrocatalysts were utilized as the cathode. Three TiO2/Ti felt electrodes with different oxide thicknesses were prepared and their characterization results were correlated with their respective electrochemical performance in terms of Faradaic efficiency , and partial current density . Cyclic voltammetry indicated a different electrocatalytic reduction process on hydrothermally treated electrodes, compared to thermally oxidized ones. Hydrothermally treated electrodes were also found to have the thickest porous anatase layer and achieved 50-75 % alanine conversion efficiencies. Optimization showed that the cell potential, reactant flow rate and the PA: NH2OH ratio were crucial parameters in determining the conversion efficiency. and were found to significantly decrease when an excess of is used and, an optimal alanine of 75 % was achieved at 2.0 V applied cell potential and 10 mL/h reactant flow rate.

Montmorillonite Cation Incorporated Synthesis of Amino acids from Methane-Ammonia and Water vapour under Primordial Conditions

A study was conducted to investigate the reaction of methane-ammonia water vapour in primordial conditions with and without the presence of green catalyst montmorillonite and cationexchanged montmorillonite, which resulted in the formation of significant number of amino acids. The study identified the resulting products through the use of chromatographic and spectroscopic techniques. The formation of lysine, aspartic acid, glycine, glutamic acid, alanine, valine and leucine was reported from the above experimental solutions.

THERMODYNAMICS OF COMPLEXATION WITH 18-CROWN-6 WITH L-CARNOSINE AND ITS CONFORMATIONAL ANALYSIS

The study of model biological "guest-host" processes between dipeptides and crown ethers allows us to understand the nature of molecular recognition in living systems. The reaction of the formation of the molecular complex of the crown ether 18-crown-6 (18K6) with L-carnosine (L-Carn) in water at T = 298.15 K was studied using isothermal titration calorimetry. The mixing heats of aqueous solutions of L-carnosine with aqueous solutions of 18-Carn-6 were determined on a TAM III titration calorimeter system (TA Instruments) equipped with a 20 mL titration cell. In the main experiment, the heat of mixing of aqueous 18K6 solution with aqueous L-Carn solution was determined. In separate experiments the heat effect of dilution of L-Carn solution in water was measured, which was taken into account in determining the thermal effect of complex formation. From the thermochemical data, the thermodynamic parameters of the molecular complex formation reaction were calculated. It was found that the presence of a histidine fragment in the L-Carn structure leads to an increase in the stability of the molecular complex L-Carn with 18K6 and to a decrease in the exothermicity of complex formation as compared to the complex formation of alanine with 18K6. Thermodynamic parameters of [L-Carn 18K6] formation reaction are similar to thermodynamics of 18K6 complex formation with histidine. Quantum-chemical calculations were performed within the framework of the electron density functional theory using the GAUSSSIAN 03 software package. The results were visualized using the ChemCraft software. We performed the conformational analysis of L-Carn within the framework of the density functional theory (version B3LYP/6-31G**) to analyze the reactivity of L-Carn structural fragments. For three stable conformers in the M062X/ aug-cc-pVTZ approximation the geometric parameters were obtained and the electron density distribution was analyzed within the framework of the QTAIM method as well as the NBO scheme. For citation: Usacheva T.R., Belova N.V., Saturina E.V., Krutova O.N., Lukanov M.M., Pavlova E.A. Thermodynamics of complexation with 18-crown-6 with l-carnosine and its conformational analysis. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2023. V. 66. N 5. P. 21-31. DOI: 10.6060/ivkkt.20236605.6781.

Quantum chemistry and metadynamics study of kinetic routes to alanine formation by CO or CO2 insertions in E- or Z-ethanimine isomers

Quantum chemistry and metadynamics study of kinetic routes to alanine formation by CO or CO2 insertions in E- or Z-ethanimine isomers

Proton-Coupled Electron Transfer in Electrochemical Alanine Formation from Pyruvic Acid: Mechanism of Catalytic Reaction at the Interface between TiO2 (101) and Water

Proton-Coupled Electron Transfer in Electrochemical Alanine Formation from Pyruvic Acid: Mechanism of Catalytic Reaction at the Interface between TiO₂ (101) and Water

Methods to Investigate the Kinetic Profile of Cysteine Desulfurases.

Biological iron-sulfur (Fe-S) clusters are essential protein prosthetic groups that promote a range of biochemical reactions. In vivo, these clusters are synthesized by specialized protein machineries involved in sulfur mobilization, cluster assembly, and cluster transfer to their target proteins. Cysteine desulfurases initiate the first step of sulfur activation and mobilization in cluster biosynthetic pathways. The reaction catalyzed by these enzymes involves the abstraction of sulfur from the amino acid L-cysteine, with concomitant formation of alanine. The presence and availability of a sulfur acceptor modulate the sulfurtransferase activity of this class of enzymes by altering their reaction profile and catalytic turnover rate. Herein, we describe two methods used to probe the reaction profile of cysteine desulfurases through quantification of alanine and sulfide production in these reactions.

Оценка анаэробной утилизации глюкозы штаммами Escherichia coli с нарушенной способностью к брожению при дыхании с внешним и внутренним акцептором электронов

The characteristics of anaerobic glucose utilization and metabolite production by recombinant Escherichia coli strains with impaired fermentation ability upon respiration with pyruvate as an internal and nitrate as an external electron acceptor have been studied. It was found that respiration processes utilizing pyruvic acid as an endogenous electron acceptor and leading to the lactate and alanine formation were capable of mutual interference. After elimination of ammonium ions from the medium, the native activity levels of respiratory lactate dehydrogenases Did and LldD in E. coli strains deficient in the mixed acid fermentation pathways can almost completely compensate for the loss of activity of respiratory alanine dehydrogenase DadA, but are insufficient to maintain the entire intracellular redox balance. The addition of nitrate ions in the medium abolished alanine production by the strains despite the availability of ammonium ions, while the functionality of respiratory reduction of endogenous pyruvate to lactate retained in the studied strains even in the presence of a strong exogenous oxidant. Respiration with external electron acceptor provoked the activation of the oxidative tricarboxylic acid cycle in the strains. Anaerobic glucose utilization by the strain with interrupted tricarboxylic acid cycle increased during nitrate respiration, but remained restricted by the excessive generation of reducing equivalents in the residual reactions of the cycle. Escherichia coli, glucose, fermentation, respiration, pyruvate, lactate, alanine, nitrate, ammonium. The work was supported by a grant from the Russian Foundation for Basic Research (project #18-04-01222).

Activation of Alternative Respiration with Internal Electron Acceptor during Anaerobic Glucose Utilization in Escherichia coli Strains with Impaired Fermentation Ability

The activation of alternative respiration with an internal electron acceptor during anaerobic glucose utilization in E. coli strains with impaired fermentation ability has been studied. It was found out that respiration processes utilizing pyruvic acid as an endogenous electron acceptor can markedly contribute to the maintenance of the anaerobic redox balance in E. coli strains deficient in mixed acid fermentation pathways. The sequential inactivation of the pathways of anaerobic dissimilation of pyruvate and impairment of the functionality of the reductive branch of the tricarboxylic acid cycle led to an increase in the contribution (from 11 to 54%) of the respiratory formation of lactic acid and alanine to the biosynthesis of the reduced products of anaerobic glucose utilization by the strains. Analysis of the enantiomeric composition of the lactic acid and alanine secreted by the strains demonstrated that D-lactate dehydrogenase (Dld), L-lactate dehydrogenase (LctD), and D-alanine dehydrogenase (DadA) participated in the biosynthesis of the respective compounds.

MTORC1 is required for expression of LRPPRC and cytochrome-c oxidase but not HIF-1α in Leigh syndrome French Canadian type patient fibroblasts.

Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-13C6]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.

Formation of alanine, α-aminobutyrate, acetate, and 2-butanol during cheese ripening by Pediococcus acidilactici FAM18098

There is limited information about the contribution of Pediococcus acidilactici, a nonstarter lactic acid bacteria, to cheese ripening and flavour development. Model Tilsit-type and Gruyère-type cheeses were produced using P. acidilactici FAM18098 as an adjunct. The adjunct did not influence the cheese manufacturing processes. The pediococcal log counts ranged from 7.0 to 8.0 cfu g−1 after 90 and 120 days of ripening. P. acidilactici produced ornithine, a result of arginine metabolism by the arginine deiminase pathway, and α-aminobutyrate and alanine while simultaneously metabolising serine and threonine. The analysis of the volatile compounds in the cheeses showed that higher acetate, 2-butanone, and 2-butanol levels and lower diacetyl levels were present in the cheeses produced with P. acidilactici than in the control cheeses. The study illustrates that P. acidilactici can influence amino acid metabolism in cheese; further, ornithine, α-aminobutyrate, and acetate can serve as indicators for the presence of this species.

Mechanisms and functions of ribosome-associated protein quality control.

The stalling of ribosomes during protein synthesis results in the production of truncated polypeptides that can have deleterious effects on cells and therefore must be eliminated. In eukaryotes, this function is carried out by a dedicated surveillance mechanism known as ribosome-associated protein quality control (RQC). The E3 ubiquitin ligase Ltn1 (listerin in mammals) plays a key part in RQC by targeting the aberrant nascent polypeptides for proteasomal degradation. Consistent with having an important protein quality control function, mutations in listerin cause neurodegeneration in mice. Ltn1/listerin is part of the multisubunit RQC complex, and recent findings have revealed that the Rqc2 subunit of this complex catalyses the formation of carboxy-terminal alanine and threonine tails (CAT tails), which are extensions of nascent chains known to either facilitate substrate ubiquitylation and targeting for degradation or induce protein aggregation. RQC, originally described for quality control on ribosomes translating cytosolic proteins, is now known to also have a role on the surfaces of the endoplasmic reticulum and mitochondria. This Review describes our current knowledge on RQC mechanisms, highlighting key features of Ltn1/listerin action that provide a paradigm for understanding how E3 ligases operate in protein quality control in general, and discusses how defects in this pathway may compromise cellular function and lead to disease.

Catalyst: Is the Amino Acid a New Frontier for Biorefineries?

Prof. Ning Yan obtained his BS and PhD degrees from Peking University in 2004 and 2009, respectively. Thereafter, he worked as a Marie-Curie research fellow at Ecole Polytechnique Federale de Lausanne, Switzerland. He joined the National University of Singapore (NUS) as an assistant professor, established the Lab of Green Catalysis in 2012, and was promoted to the position of associate professor in 2018. Yunzhu Wang received her BE degree from NUS in 2014. She joined Prof. Yan’s group in the same year and is currently pursuing her PhD. Her research interests include nanoscale metal catalysts and their applications in producing renewable nitrogen-containing chemicals.

Активация альтернативного дыхания с внутренним акцептором электронов при анаэробной утилизации глюкозы у штаммов Escherichia coli с нарушенной способностью к брожению

The activation of alternative respiration with an internal electron acceptor upon anaerobic utilization of glucose in Escherichia coli strains with the impaired fermentation ability has been studied. It was found out that the respiration processes utilizing pyruvic acid as an endogenous electron acceptor can markedly contribute to the maintenance of the anaerobic redox balance in E. coli strains deficient in mixed acid fermentation pathways. The sequential inactivation of the pathways of anaerobic dissimilation of pyruvate and impairment of the functionality of the reductive branch of the tricarboxylic acid cycle led to the increase in the contribution of the respiratory formation of lactic acid and alanine to the biosynthesis of the reduced products of anaerobic glucose utilization by the strains from 11 to 54%. The analysis of the enantiomeric composition of the lactic acid and alanine secreted by the strains demonstrated that D-lactate dehydrogenase (Dld), L-lactate dehydrogenase (LctD), and D-alanine dehydrogenase (DadA) participated in the biosynthesis of the respective compounds. Escherichia coli, glucose, fermentation, respiration, metabolic engineering, pyruvate, lactate, alanine. The work was supported by a grant from the Russian Foundation for Basic Research (Project no. 18-04-01222)

Biological Homochirality on the Earth, or in the Universe? A Selective Review

The discovery of meteoritic alpha-amino acids with significant enantiomeric excesses of the L-form has suggested that some cosmic factors could serve as the initial source for chiral imbalance of organic compounds delivered to the early Earth. The paper reviews major hypothesis considering the influence of chiral irradiation and chiral combinations of physical fields on the possible ways asymmetric synthesis and transformations of organics could take place within the solar system. They could result in a small enantiomeric imbalance of some groups of compounds. More attention is paid to the hypothesis on parity violation of weak interaction that was supposed to cause homochirality of all primary particles and a more significant homochirality of compounds directly synthesized from the latter in a plasma reactor. The first experiment with material synthesized in a plasma torch resulting from a super-high-velocity impact showed formation of alanine with the excess of L-form between 7 and 25%. The supposed conclusion is that L-amino acids could serve as a starting homochiral biomolecular pool for life to emerge all over the Universe.

A Possible Path to Prebiotic Peptides Involving Silica and Hydroxy Acid-Mediated Amide Bond Formation.

The formation of alanine and glycine oligomers in films produced by drying aqueous mixtures of lactic acid and silica nanoparticles has been studied as a model prebiotic reaction. The addition of silica results in alanine or glycine enrichment in the polymers. Oligomerization proceeds through ester-mediated peptide bond formation in an acidic and evaporative environment at temperatures as low as 85 °C. For both amino acids, the dominant species produced in the presence of silica and lactic acid are rich in amide bonds and deficient in ester linkages. At higher temperatures, glycine and alanine oligomers contain only a single hydroxy acid residue conjugated to the peptide N terminus. Similar product distributions occur with silica particles prereacted with lactic acid, which suggests the catalytic role of a functionalized surface. This work highlights the role minerals might have served in transitioning from oligomers with both ester and amide linkages (depsipeptides) to peptides in a prebiotic context.

Elevation of cytosolic Ca2+ in response to energy deficiency in plants: the general mechanism of adaptation to low oxygen stress.

Ca2+ can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca2+ release under conditions of ATP concentration decline that result in the suppression of ATPases and activation of calcium ion channels. The main signaling and metabolic consequences of Ca2+ release are considered for stressed plant cells. The signaling function includes generation and spreading of calcium waves, while the metabolic function results in the activation of particular enzymes and genes. Ca2+ is involved in the activation of glutamate decarboxylase, initiating the γ-aminobutyric acid shunt and triggering the formation of alanine, processes which play a role, in particular, in pH regulation. Ca2+ activates the transcription of several genes, e.g. of plant hemoglobin (phytoglobin, Pgb) which scavenges nitric oxide and regulates redox and energy balance through the Pgb-nitric oxide cycle. This cycle involves NADH and NADPH oxidation from the cytosolic side of mitochondria, in which Ca2+- and low pH-activated external NADH and NADPH dehydrogenases participate. Ca2+ can also activate the genes of alcohol dehydrogenase and pyruvate decarboxylase stimulating hypoxic fermentation. It is concluded that calcium is a primary factor that causes the metabolic shift under conditions of oxygen deficiency.

A theoretical quantum chemical study of alanine formation in interstellar medium

The interstellar medium, the vast space between the stars, is a rich reservoir of molecular material ranging from simple diatomic molecules to more complex, astrobiologically important molecules such as amino acids, nucleobases, and other organic species. Radical-radical and radical-neutral interaction schemes are very important for the formation of comparatively complex molecules in low temperature chemistry. An attempt has been made to explore the possibility of formation of complex organic molecules in interstellar medium, through detected interstellar molecules like CH3CN and HCOOH. The gas phase reactions are theoretically studied using quantum chemical techniques. We used the density functional theory (DFT) at the B3LYP/6-311G(d, p) level. The reaction energies, potential barrier and optimized structures of all the geometries, involved in the reaction path, has been discussed. We report the potential energy surfaces for the reactions considered in this work.