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The flexibility of aβhairpin structure known as theflapplays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequential similarity. In the apo form of the proteases, a conserved tyrosine residue in the flap region is in dynamic equilibrium between the normal and flipped states through rotation of theχ1andχ2angles. In this study, we have used apo Plm-II and BACE-1 as model systems. Independent MD simulations of Plm-II and BACE-1 remained stuck either in the normal or flipped state. Metadynamics simulations using side-chain torsion angles (χ1andχ2of tyrosine) as collective variables sampled the transition between the normal and flipped states. Qualitatively, the two states were predicted to be equally populated. The normal and flipped states were stabilised by H-bond interactions to a tryptophan residue and to the catalytic aspartate, respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine, reduced the flexibility of the flap and resulted in a flap collapse (flap loses flexibility and remains stuck in a particular state). This is in accordance with previous experimental studies, which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Our results suggest that the rotation of the tyrosine side-chain is the key movement that governs flap dynamics and opening of the binding pocket in most pepsin-like aspartic proteases.

Glatiramer acetate (GA), FDA-approved therapeutic for multiple sclerosis (MS), is a random-sized synthetic polypeptide composed of four amino acids: tyrosine, glutamic acid, alanine, and lysine. This work aims to design an efficient protocol for the separation of GA from its broth. For this purpose, the feasibility of a carbohydrate-based aqueous two-phase system (ATPS) for separation of GA from the amino acids was investigated. Initially, the binodal curve and tie-lines for the ATPS composed of maltodextrin and acetonitrile were determined. Next, preliminary screening experiments for determining optimum conditions were conducted by introducing GA and amino acids into the ATPS individually and the effect of pH and carbohydrate concentration on partition coefficient were investigated. Results revealed that glutamic acid and alanine were migrated toward the acetonitrile rich phase while GA, tyrosine, and lysin showed the opposite trend. Besides, the partitioning of amino acids was correlated with their physicochemical and structural properties. The preliminary results suggested that the system composed of maltodextrin 15 wt%, acetonitrile 35 wt%, at pH = 6 can be considered as the optimum feed. In the next step, the partitioning of GA and amino acids broth was investigated in the optimum feed. Under these conditions, the partition coefficient and selectivity of GA were obtained to be 1.17 and 62%, respectively. Additionally, circular dichroism spectroscopy results proved that the structure of GA remains unchanged during the separation steps. Finally, a recovery method was proposed where the acetonitrile was evaporated and maltodextrin was precipitated by the addition of methanol.

N-(Phosphonomethyl)glycine (glyphosate), known by the trade name Roundup®, is a broad-spectrum systemic herbicide used to kill various types of weeds. It was first synthesized in 1970 by John E. Franz, a chemist at the Monsanto agrochemical company. Glyphosate's mode of action is to inhibit a plant enzyme involved in the synthesis of some aromatic amino acids (“shikimate way”). The use of Roundup® is currently controversial, as its hazard potential has not been clarified. Glufosinate (2-Amino-4-[hydroxy(methylphosphonoyl)] butanoic acid) was discovered by German and Japanese scientists in a biological process: Species of Streptomyces bacteria produce a tripeptide that consists of two alanine residues and an amino acid that is an analogue of glutamate named phosphinothricin. Phosphinothricin was first synthesized by scientists at Hoechst (now Aventis) in the 1970s as a racemic mixture; this racemic mixture is called glufosinate. This article presents reliable and easily performed spectroscopic and (spectro)electrochemical measurements for identifying glyphosate and glufosinate.

The amino acids synthesized by the intestinal microbiota have been correlated with metabolic diseases, such as type 2 diabetes mellitus and insulin resistance; both are high incidence conditions in Mexico. However, the knowledge of the relationship of fecal amino acids with the development of both diseases in the Mexican population is scarce. The clinical study was descriptive; the study was carried out in the Antiguo Civil Hospital of Guadalajara. Samples were taken from a total of 48 participants with insulin resistance, diabetes, and a control group (n = 16 each). Anthropometric and biochemical measures were evaluated. HPLC carried out the quantification of fecal amino acids. A strong correlation between alanine and HOMA-IR (r = 0.5416) was found and between phenylalanine and HOMA-IR (r = 0.4258). Other interesting correlations were between alanine and glucose (r = 0.5854) and isoleucine and glucose (r = 0.5008). The diabetic group and the insulin-resistant group had increased fecal values of valine and isoleucine (branched-chain amino acids), which were positively correlated with the progression of both conditions. Likewise, alanine and phenylalanine can help predict the development of the disease in the Mexican population. Registry number: 037/19.

Electrochemical studies of resorcinol-based acridinedione (AD) dyes with nonfluorophoric simple amino acids, glycine, alanine, and valine, were carried out in water. AD probes are classified into photoinduced electron transfer (PET) and non-PET-based dyes, wherein the electrochemical properties and photophysical and photochemical behavior vary significantly based on the nature of substituent groups and the nature of the solute. The oxidation potential of PET dye (ADR1) to that of non-PET-based dye (ADR2) differs significantly such that the addition of amino acids results in a shift of the oxidation peak to a less positive potential and the reduction peak to a lesser negative potential. The extent of shift of oxidation and reduction potential in PET dye is more pronounced than that of non-PET dye on the addition of valine rather than glycine. The variation in the shift is attributed to the presence of an electron-donating moiety (OCH3) group in the ninth position of ADR1 dye. Consequently, the quenching of fluorescence is observed in ADR2 with non fluorophoric amino acids that are authenticated by the shift of the anodic and cathodic peaks toward a lesser positive potential. Molecular docking (MD) studies of PET and non-PET dye with amino acids portray that neither hydrophobic interactions nor electrostatic or weak interactions such as van der Waals and pi–pi interactions govern the electrochemical nature of dye on the addition of amino acids. Furthermore, the formation of a conventional hydrogen bond between dye and amino acid is established from MD studies. The existence of dye–water–amino acid competitive hydrogen-bonding interactions is presumably well-oriented throughout the aqueous phase as observed through photophysical studies which support our electrochemical investigation.

Made available in DSpace on 2022-04-29T08:35:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 The COVID-19 pandemic prompted the FDA to authorize a new nucleoside analogue, remdesivir, for emergency use in affected individuals. We examined the effects of its active metabolite, remdesivir triphosphate (RTP), on the activity of the replicative mitochondrial DNA polymerase, Pol γ. We found that while RTP is not incorporated by Pol γ into a nascent DNA strand, it remains associated with the enzyme impeding its synthetic activity and stimulating exonucleolysis. In spite of that, we found no evidence for deleterious effects of remdesivir treatment on the integrity of the mitochondrial genome in human cells in culture. Department of Chemistry Auburn University at Montgomery Department of Biochemistry and Molecular Biology Southern Illinois University School of Medicine Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho” Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho”

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry-tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies.

Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

Belactosins and hormaomycins are peptide natural products containing 3-(2-aminocyclopropyl)alanine and 3-(2-nitrocyclopropyl)alanine residues, respectively, with opposite stereoconfigurations of the cyclopropane ring. Herein we demonstrate that the heme oxygenase-like enzymes BelK and HrmI catalyze the N-oxygenation of l-lysine to generate 6-nitronorleucine. The nonheme iron enzymes BelL and HrmJ then cyclize the nitroalkane moiety to the nitrocyclopropane ring with the desired stereochemistry found in the corresponding natural products. We also show that both cyclopropanases remove the 4-proS-H of 6-nitronorleucine during the cyclization, establishing the inversion and retention of the configuration at C4 during the BelL and HrmJ reactions, respectively. This study reveals the unique strategy for stereocontrolled cyclopropane synthesis in nature.

RATIONALE The fatty acid amide oleoyl glycine (OlGly) and its more stable methylated form oleoyl alanine (OlAla) reduce naloxone-precipitated morphine withdrawal (MWD)-induced conditioned gaping (nausea) responses in rats. In addition, OlGly has been shown to reduce lithium chloride (LiCl)-induced conditioned gaping in rats and vomiting in Suncus murinus (house musk shrews). OBJECTIVES Here, we compared the potential of these fatty acid amides to maintain their anti-nausea/anti-emetic effect over a delay. The following experiments examined the potential of a wider dose range of OlGly and OlAla to interfere with (1) LiCl-induced conditioned gaping in rats and (2) LiCl-induced vomiting in shrews, when administered 20 or 70 min prior to illness. RESULTS OlAla (1, 5, 20 mg/kg) reduced LiCl-induced conditioned gaping, with OlGly only effective at the high dose (20 mg/kg), with no effect of pretreatment delay time. At the high dose of 20 mg/kg, OlGly increased passive drips during conditioning suggesting a sedative effect. In shrews, both OlGly and OlAla (1, 5 mg/kg) suppressed LiCl-induced vomiting, with no effect of pretreatment delay. OlAla more effectively suppressed vomiting, with OlAla (5 mg/kg) also increasing the latency to the first vomiting reaction. CONCLUSIONS OlAla was more effective than OlGly in reducing both LiCl-induced gaping in rats and LiCl-induced vomiting in shrews. These findings provide further evidence that these fatty acid amides may be useful treatments for nausea and vomiting, with OlAla demonstrating superior efficacy.

Ethylene (ETH) is a gaseous plant growth regulator that affects grain yield and quality in rice (Oryza sativa L.). Little is known about whether and how ETH regulates amino acid contents in milled rice. In this study, three categories of rice cultivars differing in protein content were field grown and the relationship of ethylene evolution rate (EER) and its synthetic precursor (1-aminocylopropane-1-carboxylic acid, ACC) in filling grains with amino acid biosynthesis were investigated. The results showed that rice cultivars with higher levels of ETH and ACC exhibited lower amino acid contents than the others. The EER and ACC content were highly significantly and negatively correlated with the activities of glutamate synthetase (GOGAT), aspartate transaminase (AST) and alanine transaminase (ALT) involved in amino acid biosynthesis, and the contents of essential amino acids (EAAs), non-essential amino acids (NEAAs), and total amino acids (TAAs), serine (Ser), alanine (Ala), lysine (Lys), threonine (Thr) and leucine (Leu) in milled rice. The application of ethephon (an ethylene-releasing agent) or ACC to rice panicles markedly reduced the activities of the above three enzymes and contents of amino acids, especially Ser, Ala, Lys, Thr and Leu, whereas the use of aminoethoxyvinylglycine (AVG, an inhibitor of ACC synthase) had the opposite effect. These results suggest that high ETH level negatively affects amino acid biosynthesis mainly by inhibiting the activities of GOGAT, AST, and ALT during the grain filling in rice.

Accumulating evidence suggests that amino acids are important indicators of nutritional and metabolic status. A high-resolution mass spectrometry method based on sequential window acquisition of all theoretical mass spectra acquisition was developed for the simultaneous determination of 16 amino acids in human plasma. Sample preparation by protein precipitation using a mixture of acetonitrile and formic acid was followed by a BEH Amide column. The superiority of this method was investigated by comparing it to time-of-flight scan and multiple reaction monitoring modes. The limit of detection in sequential window acquisition of all theoretical mass spectra mode for threonine, methionine, histidine, citrulline, and tryptophan is 0.1 ng on the column; for lysine and asparagine is 0.2 ng; for alanine, pyroglutamic acid, leucine, ornithine, and aspartate is 0.5 ng, for arginine is 1.0 ng; for glutamate and serine is 2.0 ng; for glutamine is 10.0 ng. This method was linear in the range 0.8-40 μg/mL for arginine, citrulline, glutamate, histidine, leucine, methionine, pyroglutamic acid, threonine, tryptophan; 2-100 μg/mL for asparagine, aspartate, lysine, ornithine, serine; and 4-200 μg/mL for alanine, glutamine with good accuracy and precision. Significantly different levels in 11 amino acids were observed between childhood and adulthood, representing the growth and development of individuals relating to the level of amino acids.

In this paper, we examined the competence of amino acids as standards for instrumental biochemical analysis. The chosen amino acids were first dissolved in various aquatic solutions and then measured in a benchtop NMR spectrometer, which is not a common choice in such analytical investigations. Analysis by mass spectrometry was used in addition. As part of these investigations, we examined and determined the stability of the amino acids ornithine, glutamic acid, alanine, glycine, proline, pyroglutamic acid, phenylalanine and trans-4-hydroxy-D-proline under critical basic and acidic pH conditions and under various other conditions. We observed that not all solutions of the amino acid standards remain stable under the given conditions and a chemical transformation takes place. Given our findings by mass spectroscopy, additional kinetic measurements were carried out with the benchtop NMR spectrometer. We discovered that pyroglutamic acid becomes unstable under basic conditions and decarboxylates to pyrrolidone.

Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.

Photochemical redox reactions of Cu(II) complexes of eight amino acid ligands (L) with nonpolar side chains have been systematically investigated in deaerated aqueous solutions. Under irradiation at 313 nm, the intramolecular carboxylate-to-Cu(II) charge transfer within Cu(II)–amino acid complexes leads to Cu(I) formation and the concomitant decomposition of amino acids. All amino acid systems studied here can produce ammonia and aldehydes except proline. For the 1:1 Cu(II) complex species (CuL), the Cu(I) quantum yields at 313 nm (ΦCu(I),CuL) vary by fivefold and in the sequence (0.10 M ionic strength at 25 °C) alanine (0.094) > valine (0.059), leucine (0.059), isoleucine (0.056), phenylalanine (0.057) > glycine (0.052) > methionine (0.032) > proline (0.019). This trend can be rationalized by considering the stability of the carbon-centered radicals and the efficient depopulation of the photoexcited state, both of which are dependent on the side-chain structure. For the 1:2 Cu(II) complex species (CuL2), the Cu(I) quantum yields exhibit a similar trend and are always less than those for CuL. The photoformation rates of ammonia, Cu(I), and aldehydes are in the ratio of 1:2.0 ± 0.2:0.7 ± 0.2, which supports the proposed mechanism. This study suggests that the direct phototransformation of Cu(II)–amino acid complexes may contribute to the bioavailable nitrogen for aquatic microorganisms and cause biological damage on cell surfaces in sunlit waters.

Despite more than 100 years of research, formation of food melanoidins from carbohydrates and amino acids in the course of the Maillard reaction is still not fully understood. Experiments with relevant precursors are commonly used to limit the pathways of the complex reaction and to elucidate the formation mechanisms of the colored end-products. Here as a simple model, methylglyoxal was incubated with l-alanine or l-lysine in aqueous solutions at 100 °C and pH 5. The reaction mixtures were analyzed for color formation, molecular weight distribution, and conversion of methylglyoxal. High-resolution mass spectrometry was used to characterize the variety of products formed. With the help of Kendrick and van Krevelen analyses, the complex data sets were investigated for common substructures and reaction patterns. This study revealed that methylglyoxal forms oligomers via aldol reaction under involvement of its prevalent reaction products such as formaldehyde, acetaldehyde, acetol, and aminoacetone with amino acids.

Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear. Here, we report how the repeats contribute to overall RNA binding. We first prepared three mutants in which R1' and/or R8' were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1' contributed more than R8', unlike Drosophila PUM. We next investigated which amino acid residues of R1' or R8' were responsible for RNA binding. With detailed analysis of the protein tertiary structure, we found a hydrophobic core in each of the repeats. We therefore mutated all hydrophobic amino residues in each core to alanine. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1' had a significant influence. In the present study, we demonstrated that the flanking R1' and R8' repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1'-R1 interactions may stabilize the whole hPUM structure.

The metabolic needs for postnatal growth of the human nervous system are vast. Recessive loss-of-function mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms in neurons required for neuronal growth and survival. These metabolic processes include neuronal alanine synthesis and anaplerosis, the replenishment of tricarboxylic acid (TCA) cycle intermediates. We performed metabolomics across postnatal development in Gpt2-null mouse brain to identify the trajectory of dysregulated metabolic pathways: alterations in alanine occur earliest; followed by reduced TCA cycle intermediates and reduced pyruvate; followed by elevations in glycolytic intermediates and amino acids. Neuron-specific deletion of GPT2 in mice is sufficient to cause motor abnormalities and death pre-weaning, a phenotype identical to the germline Gpt2-null mouse. Alanine biosynthesis is profoundly impeded in Gpt2-null neurons. Exogenous alanine is necessary for Gpt2-null neuronal survival in vitro but is not needed for Gpt2-null astrocytes. Dietary alanine supplementation in Gpt2-null mice enhances animal survival and improves the metabolic profile of Gpt2-null brain but does not alone appear to correct motor function. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons in vivo. We also observe selective death of lower motor neurons in vivo with worsening motor behavior with age. In conclusion, these studies of the pathophysiology of GPT2 Deficiency have identified metabolic mechanisms that are required for neuronal growth and that potentially underlie selective neuronal vulnerabilities in motor neurons.

Background We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unraveling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells. Methods 1H NMR spectroscopy of tumor tissues from meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using 13C/15N glutamine tracer, in 5 patient-derived meningioma cells. Results Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75%, and 33%, respectively, with alanine and glutamine levels being statistically significant (P ≤ .02). 13C/15N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells. Conclusion Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) is very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Aims The aim of this article is to study the functional features of Penicillium oxalicum transcriptional activator XlnR. Methods and results The yeast reporter system was used to identify transcriptional activation domain of XlnR in P. oxalicum. The expression cassette was introduced into the xlnR locus of P. oxalicum by homologous recombination. In this study, several putative structural domains in P. oxalicum XlnR were predicted by bioinformatics analysis, and the transcriptional activation domain (351-694 region) was identified in XlnR relying on reporter gene system in yeast. In addition, the amino acid at XlnR 871 site (alanine) located in the regulatory region could influence the regulatory activity of XlnR directly. When the alanine at XlnR 871 site was replaced by stronger hydrophobic amino acid (e.g. valine or isoleucine), the regulatory activity will be greatly improved, especially for the regulation of hemicellulase genes expression. When alanine at XlnR 871 site was mutated to a hydrophilic amino acid (e.g. aspartic acid or arginine), the regulatory activity of XlnR will be reduced. Conclusions The 351-694 region of P. oxalicum XlnR was identified as transcriptional activation domain, and the regulatory activity of XlnR was greatly influenced by hydrophobicity of amino acid at 871 site of XlnR in P. oxalicum. Significance and impact of the study The results will provide an effective target site to regulate the activity of XlnR and improve cellulase production of P. oxalicum.

Plantaricin Q7 is a bacteriocin produced by Lactobacillus plantarum Q7 with food preservation potential. Low yield is one of the bottlenecks of the wide application of plantaricin Q7. Nontargeted metabolomics was performed to reveal the mechanism of plantaricin Q7 biosynthesis. The results showed that the composition and abundance of intracellular metabolites varied significantly at key time points of plantaricin Q7 synthesis. Differential metabolic pathways were purine metabolism; pyrimidine metabolism; alanine, aspartate, and glutamate metabolism; amino acid biosynthesis; aminoacyl-tRNA biosynthesis; and ABC transporters. Differential metabolites were xanthine, deoxyadenosine, uracil, 5-methylcytosine, α-ketoglutarate, γ-aminobutyric acid, glutamate, glutamine, and tryptophan. Based on metabolomics information, the putative metabolic synthesis pathway of plantaricin Q7 was proposed. Glutamine, glutamate, and 5-methylcytosine could be critical metabolites and simulate plantaricin Q7 biosynthesis significantly (P < 0.05). Bacteriocin production was investigated by comparative metabolomics in this report, which could help to achieve higher plantaricin Q7 yield by metabolic regulation.

The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources. This process is mainly governed by the global regulator catabolite control protein A (CcpA), which can recognize and bind to multiple target genes that are widely distributed in metabolic pathways. Although the DNA-binding domain of CcpA has been predicted, the influence of key amino acids on target gene recognition and binding has yet to be uncovered. In this study, the impact of Lys31, Ile42 and Leu56 on in vitro protein–DNA interactions and in vivo carbon source selective utilization was investigated. The results showed that alanine substitution of Lys31 and Ile42, located within the 3rd helices of the DNA-binding domain, significantly weakened the binding strength between CcpA and target genes. These mutations also lead to alleviated repression of xylose utilization in the presence of glucose. On the other hand, the Leu56Arg mutant in the 4th helices exhibited enhanced binding affinity compared with that of the wild-type one. When this mutant was used to replace the native one in B. licheniformis cells, the selective utilization of glucose over xylose increased. This study provides a new strategy for understanding the relationship between the function and structure of regulatory proteins. This study also used a new strategy was used to regulate carbon source utilization beyond CCR engineering.

Selective modifications of peptides and proteins have emerged as a promising strategy to develop novel mechanistic probes and prepare compounds with translational potentials. Here, we report alanine carbastannatranes AlaSn as a universal synthon in various C-C and C-heteroatom bond-forming reactions. These reagents are compatible with peptide manipulation techniques and can undergo chemoselective conjugation in minutes when promoted by Pd(0). Despite their increased nucleophilicity and propensity to transfer the alkyl group, C(sp3)-C(sp2) coupling with AlaSn can be accomplished at room temperature under buffered conditions (pH 6.5-8.5). We also show that AlaSn can be easily transformed into several canonical L- and D-amino acids in arylation, acylation, and etherification reactions. Furthermore, AlaSn can partake in macrocyclizations exemplified by the synthesis of medium size cyclic peptides with various topologies. Taken together, metalated alanine AlaSn demonstrates unparalleled scope and represents a new type of umpolung reagents suitable for structure-activity relationship studies and peptide diversification.

Sorption to mineral surfaces is a key process that protects amino acids from oxidation and aids their polymerization into complex biomolecules. Sorption of neutral amino acids is driven by a combination of intermolecular forces, commonly through hydrogen bonds with surface functional groups. Substitutions of heavy isotopes are known to influence the strength of intermolecular interactions, but global C isotope fractionation associated with sorption is small ( 2‰) was observed for specific positions within a molecule during chromatographic separation, indicating that fractionation during sorption is likely more significant at positions that interact with a surface. We used quantitative isotopic 13C NMR to measure the relative difference in position-specific C isotopic distributions within glycine, l -alanine, l -serine, l -leucine, and l -phenylalanine sorbed to an ice surface versus in an aqueous solution. Isotopic differences up to 8.5‰ at functional sites were observed between sorbed and free amino acids, suggesting that sorption can alter primary isotopic patterns associated with their synthesis. Further, postion-specific isotope patterns appear to reflect the orientation of amino acids on the ice surface. As a result, position-specific isotope analysis may provide further understanding of mineral-mediated polymerization reactions.

Amino acid neurotransmitters are responsible for many physiological and pathological processes, and their cerebral concentrations respond to external influences such as the light-dark cycle and to the synthesis, release, and recapture rhythms and form part of the biochemical relationships derived from excitatory-inhibitory (E/I), glutamine-glutamate sum (GLX), glutamatergic processing (glutamine-glutamate ratio) and excitotoxic indexes. The changes in these variables during a 24-h period (1 day) are important because they allow organisms to adapt to external stimuli and form part of physiological processes. Under pathological conditions, the damage produced by acute events may depend on diurnal variations. Therefore, it is important to analyze the extracellular levels of amino acids as well as the above-mentioned indexes over a 24-h period. We focused on determining the cerebrospinal fluid levels of different amino acid neurotransmitters, and the E/I, GLX, glutamatergic processing and excitotoxic indexes, determined by microdialysis over a 24-h cycle. Our results showed significant changes during the 24-h light/dark cycle. Specifically, we found increments in the levels of glutamate (325%), GABA (550%), glutamine (300%), glycine (194%), alanine (304%) and the GLX index (263%) throughout the day, and the maximum levels of glutamate, glutamine, glycine, and alanine were obtained during the last period of the light period. In conclusion, the concentration of some amino acid neurotransmitters and the GLX index show variations depending on the light-dark cycle.

The purpose of this study was to evaluate the impacts of two amino acids that contrast in their composition: alanine as a neutral amino acid (represent regular amino acid) and methionine (represent sulfur-containing amino acids) on nitrogen (N) dynamics in acidic subtropical forest soil. 15 N-labeled alanine and methionine were added into forest soil to carry out subsequent incubation experiments for 72 h under 60% and 90% of water-holding capacities (WHC) to investigate their potential impacts. The results showed that under 60% of WHC, alanine and methionine added treatment significantly increased the net ammonification rate, except at the 5th h for alanine addition due to alanine immobilized then to prime the soil N ammonification. Alanine addition induced NO3−-N immobilization before 24 h and then nitrification occurred dominantly between 24 and 72 h. However, methionine addition resulted in a lower NO3−-N compared to control indicating that nitrification was likely inhibited. Only methionine added treatment enhanced significantly the net N2O emission rate as compared to that in the control at the 72nd h due to nitrifier denitrification possibly. Under 90% of WHC, the net ammonification rate was significantly higher in the alanine added treatment than in the control, but lower in the methionine added treatment at the 5th h. Amino acids addition significantly decreased the net nitrification rate at the 5th h, but greatly increased the net N2O emissions rate at the 24th h and 72nd h compared to control. Overall, more than 96% of NH4+-N, NO3−-N, and N2O emissions were recorded from soil native N rather than from the added amino acids. Contrasting effects of amino acids should be considered when studying the mechanisms of soil organic N in contributing to the N transformation in subtropical forest soil.

Antimicrobial peptides (AMPs) are persisting in all living organisms as evolutionarily conserved inherent components of the innate immune system. Due to relatively lower microbial drug resistance, AMPs can act as efficient small molecule based drugs. Among which, Ocellatin-1 (O1) AMP, extracted from South American frog Leptodactylus ocellatus poses greater restorative competence against pathogens. Analyses of the key amino acid residues in O1 AMP have suggested vital properties linked to the maintenance of its structural stability and the functional activity. To distinguish the contribution of side chains in each residue that facilitates its pharmacodynamics characteristics, we combined molecular simulation and computational Alanine Scanning Mutagenesis (cASM). This approach explores the alanine substituted analogues in comparison with native O1 peptide, we identified at sequence analysis, conformational sampling and membrane simulation. We observed isoleucine at 5th position and leucine at 14th position to be crucial for the therapeutic scaffold of O1 AMP. In conclusion, the role of individual residues in O1 AMP scaffold remains rationale behind maintaining its structural integrity and therefore its therapeutic potential; this therapeutic scaffold will need to be considered in future studies of membranolytic O1 peptide-based drug discovery.

The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect cell function and metabolism, but the differential effects of EPA and DHA are not known. In a randomized, controlled, double-blind, crossover study, we assessed the effects of 10-week supplementation with EPA-only and DHA-only (3 g/d), relative to a 4-week lead-in phase of high oleic acid sunflower oil (3 g/day, defined as baseline), on fasting serum metabolites in 21 subjects (9 men and 12 post-menopausal women) with chronic inflammation and some characteristics of metabolic syndrome. Relative to baseline, EPA significantly lowered the tricarboxylic acid (TCA) cycle intermediates fumarate and α-ketoglutarate and increased glucuronate, UDP-glucuronate, and non-esterified DHA. DHA significantly lowered the TCA cycle intermediates pyruvate, citrate, isocitrate, fumarate, α-ketoglutarate, and malate, and increased succinate and glucuronate. Pathway analysis showed that both EPA and DHA significantly affected the TCA cycle, the interconversion of pentose and glucuronate, and alanine, and aspartate and glutamate pathways (FDR

The cyanobacterial non-protein amino acid α-amino-β-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system xc_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.

Metal binding affinities play a vital role in medicinal, biological, and industrial applications. In particular, metal cation-amino acid (AA) interactions contribute to protein stability such that analyzing analogous prototypical interactions is important. Here, we present a full description of the interactions of sodium cations (Na+) and six aliphatic amino acids (AA), where AA = glycine (Gly), alanine (Ala), homoalanine (hAla), valine (Val), leucine (Leu), and isoleucine (Ile). Experimentally, these interactions are evaluated using threshold collision-induced dissociation carried out in a guided ion beam tandem mass spectrometer, allowing for the determination of the kinetic-energy-dependent behavior of Na+-AA dissociation. Analysis of these dissociation cross sections, after accounting for multiple ion-molecule collisions, internal energy of reactant ions, and unimolecular decay rates, allows the determination of absolute Na+-AA bond dissociation energies (BDEs) in kJ/mol of Gly (164.0), Ala (166.9), hAla (167.9), Val (172.7), Leu (173.7), and Ile (174.6). These are favorably compared to quantum chemical calculations conducted at the B3LYP, B3P86, MP2(full), B3LYP-GD3BJ, and M06-2X levels of theory. Our combination of structural and energetic analyses provides information regarding the specific factors responsible for Na+ interactions with amino acids. Specifically, we find that the BDEs increase linearly with increasing polarizability of the amino acid.

The motivation for this study is the potential of environmental friendly amino acids such as alanine, histidine, phenylalanine and valine to be examined as inhibitors on methane (95%)—propane (5%) hydrate formation together with the hydrodynamic examination of three different flows in hydrate formation process. The outcomes indicated that valine and alanine behave as promoters while histidine and phenylalanine behave as inhibitors. From hydrodynamic perception radial flow shows less induction time values and better rate of hydrate formation due to better gas liquid contact compared to mixed flow experiments.

The Pd‐catalyzed stereoselective sp3 C−H activation and arylation of various carboxamides of amino acids with iodobiaryls are reported. The synthesis of biaryl motif installed (DL‐), L‐ and D‐amino acid derivatives including norvaline, phenylalanine, leucine, norleucine, 2‐aminooctanoic acid (with anti stereochemistry), alanine and aminoalkanoic acids has been described.

Cartilaginous fish are the most primitive extant species with MHC molecules. Using the nurse shark, the current study is, to the best of our knowledge, the first to present a peptide-loaded MHC class I (pMHC-I) structure for this class of animals. The overall structure was found to be similar between cartilaginous fish and bony animals, showing remarkable conservation of interactions between the three pMHC-I components H chain, β2-microglobulin (β2-m), and peptide ligand. In most previous studies, relatively little attention was given to the details of binding between the H chain and β2-m, and our study provides important new insights. A pronounced conserved feature involves the insertion of a large β2-m F56+W60 hydrophobic knob into a pleat of the β-sheet floor of the H chain α1α2 domain, with the knob being surrounded by conserved residues. Another conserved feature is a hydrogen bond between β2-m Y10 and a proline in the α3 domain of the H chain. By alanine substitution analysis, we found that the conserved β2-m residues Y10, D53, F56, and W60—each binding the H chain—are required for stable pMHC-I complex formation. For the β2-m residues Y10 and F56, such observations have not been reported before. The combined data indicate that for stable pMHC-I complex formation β2-m should not only bind the α1α2 domain but also the α3 domain. Knowing the conserved structural features of pMHC-I should be helpful for future elucidations of the mechanisms of pMHC-I complex formation and peptide editing.

Searching for new drugs is still a challenge for science, mainly because of civilization development and globalization which promote the rapid spread of diseases, which is particularly dangerous in the case of infectious ones. Moreover, readily available already known antibiotics are often overused or misused, possibly contributing to the increase in the number of multidrug-resistant microorganisms. A consequence of this is the need for new structures of potential drugs. One of them is a benzoxazole moiety, a basic skeleton of a group of fluorescent heterocyclic compounds already widely used in chemistry, industry, and medicine, which is also present in naturally occurring biologically active compounds. Moreover, synthetic benzoxazoles are also biologically active. Considering all of that, a large group of non-proteinogenic amino acids based on 3-(2-benzoxazol-5-yl)alanine skeleton was studied in search for new antimicrobial and anticancer agents. Screening tests revealed that antibacterial potential of 41 compounds studied is not very high; however, they are selective acting only against Gram-positive bacteria (B. subtilis). Moreover, almost half of the studied compounds have antifungal properties, also against pathogens (C. albicans). Most of studied compounds are toxic to both normal and cancer cells. However, in a few cases, toxicity to normal cells is much lower than for cancer cells indicating these compounds as future anticancer agents. The research carried out on such a large group of compounds allowed to establish a structure–activity relationship which enables to select candidates for further modifications, necessary to improve their biological activity and obtain a new lead structure with potential for therapeutic use.

Background Epithelial ovarian cancer (EOC) is a common gynecological cancer with high mortality rates. The main objective of this study was to investigate the serum amino acid and organic acid profiles to distinguish key metabolites for screening EOC patients. Methods In total, 39 patients with EOC and 31 healthy controls were selected as the training set. Serum amino acid and organic acid profiles were determined using the targeted metabolomics approach. Metabolite profiles were processed via multivariate analysis to identify potential metabolites and construct a metabolic network. Finally, a test dataset derived from 29 patients and 28 healthy controls was constructed to validate the potential metabolites. Results Distinct amino acid and organic acid profiles were obtained between EOC and healthy control groups. Methionine, glutamine, asparagine, glutamic acid and glycolic acid were identified as potential metabolites to distinguish EOC from control samples. The areas under the curve for methionine, glutamine, asparagine, glutamic acid and glycolic acid were 0.775, 0 778, 0.955, 0.874 and 0.897, respectively, in the validation study. Metabolic network analysis of the training set indicated key roles of alanine, aspartate and glutamate metabolism as well as D-glutamine and D-glutamate metabolism in the pathogenesis of EOC. Conclusions Amino acid and organic acid profiles may serve as potential screening tools for EOC. Data from this study provide useful information to bridge gaps in the understanding of the amino acid and organic acid alterations associated with epithelial ovarian cancer.

Objective: The present work deals with the concerned study to determine the influence of variable season on amino acid evaluation from 90 bovine milk collected (March 2018 to February 2019) samples belongs to four tehsils of Singrauli district. Methods: An aliquot of hydrolysate of collected bovine milk samples were injected into the column (Shim-pack ISC-07/S1504 Na) of the higher performance liquid chromatography-based amino acid analyzer. Results: Most abundant amino acids were glutamic acid followed by proline and leucine. Average amino acid content comparison, results, in the rainy season, maximum amino acid content was in Deosar and minimum in Singrauli, In winter amino acid is higher in Waidhan and minimum in Deosar and maximum amino acid content was recorded intended for Waidhan and minimum for Deosar in summer. Concentration of amino acid in cow milk was found to be in order of glycine

d-Amino acid oxidase (DAAO) enzymes bind a range of d-amino acids with variable affinity. As such, the design of selective DAAO-based enzymatic biosensors remains a challenge for real-world biosensor application. Herein, a methodology for developing biosensors with varying substrate selectivity is presented. First, we address DAAO-based biosensor selectivity toward d-serine by introducing point mutations into DAAO using rational design. Next, the wild-type yeast DAAO (

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Aim: The main objective of this study is to compare and analyse human prostasomes amino acid content variation in normal men semen samples and infertile men semen samples for identification of clinical relevance. Materials and methods: Semen samples were collected from normal men (N=32) and from infertile men (N=32) and by following the standard world health organisation protocol semen analysis was done. Amino acid quantification was done by using amino acid analyzer. Prostasomes were separated from spermatozoa and seminal plasma by using centrifugation technique at 95000 RPM for 90 mins. Results: Independent sample T-test was carried out and shows that proline and alanine amino acids concentration (p

Experimental studies have indicated that electrophilic mercury forms (e.g., methylmercury, MeHg+) can accelerate the breakage of selenocysteine in vitro. Particularly, in 2009, Khan et al. (Environ. Toxicol. Chem. 2009, 28, 1567-1577) proposed a mechanism for the degradation of a free methylmercury selenocysteinate complex that was theoretically supported by Asaduzzaman et al. (Inorg. Chem. 2010, 50, 2366-2372). However, little is known about the fate of methylmercury selenocysteinate complexes embedded in an enzyme, especially in conditions of oxidative stress in which methylmercury target enzymes operate. Here, an accurate computational study on molecular models (level of theory: COSMO-ZORA-BLYP-D3(BJ)/TZ2P) was carried out to investigate the formation of dehydroalanine (Dha) in selenoenzymes, which irreversibly impairs their function. Methylselenocysteine as well as methylcysteine and methyltellurocysteine were included to gain insight on the peculiar behavior of selenium. Dha forms in a two-step process, i.e., the oxidation of the chalcogen nucleus followed by a syn-elimination leading to the alkene and the chalcogenic acid. The effect of an excess of hydrogen peroxide, which may lead to the formation of chalcogenones before the elimination, and of MeHg+, a severe toxicant targeting selenoproteins, which leads to the formation of methylmercury selenocysteinate, are also studied with the aim of assessing whether these pathological conditions facilitate the formation of Dha. Indeed, elimination occurs after chalcogen oxidation and MeHg+ facilitates the process. These results indicate a possible mechanism of toxicity of MeHg+ in selenoproteins.

Objective To evaluate the difference between Baishao (Paeoniae Radix Alba, PRA) and Chishao (Paeoniae Radix Rubra, PRR) from different regions based on the characteristic spectra of amino acids (AAs). Methods Fingerprints of the 21 standard AAs were established using O-phthalaldehyde-9-fluorenylmethyl chloroformate (OPA-FMOC) pre-column derivation method. The AA components in PRA and PRR were characterized qualitatively and quantitatively, and different AAs were screened using pattern recognition technology. Results Twelve AAs were identified in both PRA and PRR. Meanwhile, aspartic acid, glutamic acid, arginine, alanine, and gamma-aminobutyric acid were screened as characteristic components, and their concentrations could effectively distinguish PRA from PRR. Conclusion The established characterization method, which is based on the characteristic spectra of AAs, is accurate, efficient, and sensitive and can effectively distinguish between PRA and PRR from different producing areas, thus providing a reference for the overall characterization and evaluation of Chinese medicinal materials.

As life expectancy increases, an increasing older population may require surgery with perioperative nutritional management. While little is known about the combined effect of age and stress on amino acid metabolism during enteral nutrition, we hypothesized that blood amino acid bioavailability may be influenced not only by the characteristics of the ingested protein but also by intestinal ageing and splanchnic sequestration of amino acids. Plasma amino acid kinetics were thus evaluated in aged and adult rats receiving continuous enteral nutrition before and after standardized surgical stress.Sixteen 5-month-old and sixteen 21-month-old male rats were used. After a gastrostomy, the insertion of a jugular vein catheter and a one-week recovery, the animals were enterally fed with commercially available formulas containing whole milk proteins or a whey hydrolysate for 24 h before (healthy state) and 18 h after a standardized laparotomy (surgical stress). Data were analyzed by 3-factor ANOVA.In all rats, enteral nutrition was associated with a marked increase in plasma alanine, threonine, lysine and proline (+50 to +150 μmol/L; p 0.001), and a decrease in glycine (≈-80 μmol/L; p 0.01). For most amino acids, their availability depended first on the amino acid composition of each protein and second on surgical stress. Aging was only associated with higher tyrosine and threonine availability (p 0.001). There was only limited statistical interaction between age and surgical stress.In rats, plasma amino acid availability during continuous enteral nutrition is determined by the nature of the protein source and the occurrence of stress. The effects of aging on plasma amino acid availability seem very limited. Commonly used formulas therefore appear to be as suitable for elderly patients as for adult patients.

In this study, some biochemical data were evaluated in the blood serum of patients with angina pectoris, their ages ranged from (40 to 49) years, and the results were compared with healthy people. Cholesterol and serum enzymatic activity of Aspartate amino transferase AST; Alanine amino transferase ALT; Creatin Kinase Isoenzyme was estimated. Results can be summarized as follow A significant increment (p

D-alanine (D-Ala) and several other D-amino acids (D-AAs), unusual amino acids present in mammals, act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized D-serine in animals, D-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce D-Ala and other D-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their D-AA secretion in vitro, focusing on D-Ala. Serial dilutions of intestinal content from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial D-AA secretion, combining a D-amino acid oxidase-based enzymatic assay for rapid assessment of overall D-AA amount, followed by chiral LC-MS/MS to quantify individual D-AAs, revealing 19 out of the 38 isolated strains as D-AA producers. LC-MS/MS analysis of the eight top D-AA producers showed high levels of D-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from enzymatic assay and LC-MS/MS analysis aligned well, LC-MS/MS further revealed the existence of D-glutamate and D-aspartate, which are poor substrates for enzymatic assay. We observed large inter- and intra-species variation of D-AA secretion profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.

Strain designated DB0510T was isolated from an automobile evaporator core collected in Korea. Cells are gram-stain-positive, aerobic, and coccoid. The strain grew at 15–45 ℃, pH 5.0–8.5 and 0–8.0% (w/v) NaCl. Growth occurs on R2A, trypticase soy agar, Luria–Bertani agar, and nutrient agar. Phylogenetic analysis showed that the strain belongs to the family Dermacoccaceae and strain DB0510T was distinctly separated from validly named genera of this family. Signature nucleotides in 16S rRNA gene sequence revealed that the strain contained the Dermacoccaceae family-specific 16S rRNA signature nucleotides patterns. The major fatty acids were C17:0 and C17:1 cis-9. The only menaquinone was MK-8 (H4). The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, and two unidentified lipids. The diagnostic cell-wall amino acid at position 3 of the peptide subunit was found to be lysine. The cell-wall peptidoglycan contained alanine, aspartic acid, glutamic acid, glycine, lysine, and serine and thus the peptidoglycan type was concluded to be of A4α type with Lys-Gly-Ser-Asp interpeptide bridge. The genome size was 3.49 Mbp and G + C content of the genome DNA was 69.4 mol%. On the basis of the phenotypic, genomic, and chemotaxonomic characteristics, strain DB0510T is considered to represent a novel genus and species within the family Dermacoccaceae, for which the name Metallococcus carri gen. nov., sp. nov. is proposed. The type strain of Metallococcus carri is DB0510T (= KACC 19663 T = NBRC 113349 T).

A series of dimeric gemini surfactants on the study of DL-alanine (Ala) with ninhydrin reaction was carried out using the UV–vis spectroscopic technique. A study has been made under a pseudo 1st-order experimental situation by fixing ninhydrin ≥ 10 times over Ala. Rate constants (kψ) on kinetics of Ala with ninhydrin reaction were determined by varying pH, temperatures, concentrations of ninhydrin, and Ala and gemini surfactants. The outcomes of the present study disclose that the experiment obeys the respective first and fractional order of rate paths in [Ala] and [ninhydrin]. The role of temperature on kψ was employed to calculate various thermodynamic parameters. The infrequent role of gemini surfactants was observed upon addition of different concentrations to reaction. The observed enhancement in kψ values at different surfactant concentrations could be deduced by the pseudo-phase model. From experimental results, different constants and parameters including micelle-substrate-binding constant (KA), micelle-ninhydrin-binding constant (KB), and rate constant in micelle (km) were calculated.

Natural rubber (NR) contains cis-1,4-polyisoprene and many kinds of non-rubber components, e.g., proteins, lipids and phospholipids, which are believed to affect the properties of NR. Oxidative degradation is one of the problems for rubber performance, which shorten the service life of the product. To overcome this problem, the influence of fatty acids and proteins on the cure properties, heat-ageing behaviours, and ozone resistance of NR vulcanizates were investigated. The peroxide vulcanization was chosen to avoid the effect of essential fatty acid and proteins. The purified NR, deproteinized NR (DPNR) and lipids-removed NR (LRNR) were mixed with model fatty acid (stearic acid) and amino acid (alanine). It was found that the cure behaviour of these mixed samples showed almost the same trend. After removing proteins, the rate of oxidative degradation was faster than that of the lipid-removal samples. Lower lipid content would result in less oxidative degradation of the rubber. These findings can infer that the endogenous proteins in NR act as natural antioxidants, while the endogenous lipids are pro-oxidants.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP’s unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

2-aminoethylphosphonate:pyruvate aminotransferase (AEPT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that mediates the first step in the AEP degradation pathway. It catalyzes the transamination of 2-aminoethylphosphonate (AEP) with pyruvate to phosphonoacetaldehyde and l -alanine respectively. Although the enzyme is widely present in microorganisms, there are few reports on the structure and function of AEPT to date. Here we report the crystal structure of AEPT from Pseudomonas aeruginosa PAO1 (PaAEPT) to 2.35 A resolution in the absence of the PLP cofactor. PaAEPT crystallizes in space group P21212 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis shows that PaAEPT forms a stable dimer in solution. Our work provides a valuable starting point for further functional and mechanistic studies of the AEP degradation pathway.