Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH4+) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug-resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d-alanine-d-alanine ligase (BpDdl), required for the ATP-assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+d-Ala-d-Ala), and comparisons with orthologues enzymes suggest a ligand-induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow-up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches.

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

Antibiotic resistance poses an increasing threat to global health. To tackle this problem, the identification of principal reservoirs of antibiotic resistance genes (ARGs) plus an understanding of drivers for their evolutionary selection are important. During a PCR-based screen of ARGs associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a D-alanine-D-alanine ligase (ddl6 and ddl7) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to D-cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6. A SNP was found to be responsible for this difference in resistance phenotype between the two ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and D-cycloserine binding pockets of Ddl.

T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation.

Supplementing β-alanine (BA) improves exercise performance in efforts that are highly dependent on anaerobic glycolysis. As it has not yet been established whether it relates to climbing, the current study aimed to investigate the effects of BA on climbing-specific performance.

The determination of the folding dynamics of polypeptides and proteins is critical in characterizing their functions in biological systems. Numerous computational models and methods have been developed for studying structure formation at the atomic level. Due to its small size and simple structure, deca-alanine is used as a model system in molecular dynamics (MD) simulations. The free energy of unfolding in vacuum has been studied extensively using the end-to-end distance of the peptide as the reaction coordinate. However, few studies have been conducted in the presence of explicit solvent. Previous results show a significant decrease in the free energy of extended conformations in water, but the α-helical state is still notably favored over the extended state. Although sufficient in vacuum, we show that end-to-end distance is incapable of capturing the full complexity of deca-alanine folding in water. Using α-helical content as a second reaction coordinate, we deduce a more descriptive free-energy landscape, one which reveals a second energy minimum in the extended conformations that is of comparable free energy to the α-helical state. Equilibrium simulations demonstrate the relative stability of the extended and α-helical states in water as well as the transition between the two states. This work reveals both the necessity and challenge of determining a proper reaction coordinate to fully characterize a given process.

Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene qsd1 from wild barley, which encodes an alanine aminotransferase (AlaAT). The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley.

The Primary Hyperoxalurias (PH) are rare disorders of metabolism leading to excessive endogenous synthesis of oxalate and recurring calcium oxalate kidney stones. Alanine glyoxylate aminotransferase (AGT), deficient in PH type 1, is a key enzyme in limiting glyoxylate oxidation to oxalate. The affinity of AGT for its co-substrate, alanine, is low suggesting that its metabolic activity could be sub-optimal in vivo. To test this hypothesis, we examined the effect of L-alanine supplementation on oxalate synthesis in cell culture and in mouse models of Primary Hyperoxaluria Type 1 (Agxt KO), Type 2 (Grhpr KO) and in wild-type mice. Our results demonstrated that increasing L-alanine in cells decreased synthesis of oxalate and increased viability of cells expressing GO and AGT when incubated with glycolate. In both wild type and Grhpr KO male and female mice, supplementation with 10% dietary L-alanine significantly decreased urinary oxalate excretion ~30% compared to baseline levels. This study demonstrates that increasing the availability of L-alanine can increase the metabolic efficiency of AGT and reduce oxalate synthesis.

The skeletal muscle is the largest organ in the human body, depositing energy as protein/amino acids, which are degraded in catabolic conditions such as fasting. Alanine is synthesized and secreted from the skeletal muscle that is used as substrates of gluconeogenesis in the liver. During fasting, the expression of PGC-1α, a transcriptional coactivator of nuclear receptors, is increased in the liver and regulates gluconeogenesis. In the present study, we observed increased mRNA expression of PGC-1α and alanine aminotransferase 2 (ALT2) in the skeletal muscle during fasting. In C2C12 myoblast cells overexpressing PGC-1α, ALT2 expression was increased concomitant with an increased alanine level in the cells and medium. In addition, PGC-1α, along with nuclear receptor ERR, dose-dependently enhanced the ALT2 promoter activity in reporter assay using C2C12 cells. In the absence of glucose in the culture medium, mRNA levels of PGC-1α and ALT2 increased. Endogenous PGC-1α knockdown in C2C12 cells reduced ALT2 gene expression level, induced by the no-glucose medium. Taken together, in the skeletal muscle, PGC-1α activates ALT2 gene expression, and alanine production may play roles in adaptation to fasting.

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) has hampered treatments for tuberculosis, which consequently now require novel agents to overcome such drug resistance. The genetically stable D-alanine-D-alanine ligase A (DdlA) has been deemed as an excellent therapeutic target for tuberculosis. In the present study, a competitive inhibitor (IMB-0283) of DdlA was obtained via high-throughput screening. The minimum inhibitory concentrations (MIC) of IMB-0283 for the standard and clinical drug-resistant Mtb strains ranged from 0.25 to 4.00 μg/mL, whereas the conventional inhibitor of DdlA, D-cycloserine (DCS), only inhibited the growth of the standard Mtb strain at 16 μg/mL. The lethal effect of IMB-0283 on Mtb was found to act intracellularly in a DdlA-dependent manner. Specifically, IMB-0283 prevented the synthesis of neonatal cell walls but did not damage mature cell walls. Compared with those of DCS, IMB-0283 exhibited lower cytotoxicity and a higher selective index (SI). At the same dosages of treatment, IMB-0283 reduced bacterial load (log CFU/mL) in an acute animal model from 5.58 to 4.40, while DCS did not yield any such treatment efficacy. Taken together, the lower cytotoxicity and more efficacious in vivo activity of IMB-0283 suggest that it is a promising lead compound for antituberculosis drug development.

Acinetobacter baumannii is an opportunistic Gram-negative bacterium which is a common cause of hospital-acquired infections. Numerous antibiotic-resistant strains exist, emphasizing the need for the development of new antimicrobials. Alanine racemase (Alr) is a pyridoxal 5'-phosphate dependent enzyme that is responsible for racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall, its inhibition is lethal to prokaryotes, making it an excellent antibiotic drug target. The crystal structure of A. baumannii alanine racemase (AlrAba) from the highly antibiotic-resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of AlrAba with alanine racemases from closely related bacteria demonstrates a conserved overall fold. The substrate entryway and active site of the enzymes were shown to be highly conserved. The structure of AlrAba will provide the template required for future structure-based drug-design studies.

Alanine scanning mutagenesis of protein-protein interfacial residues can be applied to a wide variety of protein complexes to understand the structural and energetic characteristics of the hot-spots. Binding free energies have been estimated with reasonable accuracy with empirical methods, such as Molecular Mechanics/Poisson-Boltzmann surface area (MM-PBSA), and with more rigorous computational approaches like Free Energy Perturbation (FEP) and Thermodynamic Integration (TI). The main objective of this work is the development of an improved methodological approach, with less computational cost, that predicts accurately differences in binding free energies between the wild-type and alanine mutated complexes (DeltaDeltaG(binding)). The method was applied to three complexes, and a mean unsigned error of 0.80 kcal/mol was obtained in a set of 46 mutations. The computational method presented here achieved an overall success rate of 80% and an 82% success rate in residues for which alanine mutation causes an increase in the binding free energy > 2.0 kcal/mol (warm- and hot-spots). This fully atomistic computational methodological approach consists in a computational Molecular Dynamics simulation protocol performed in a continuum medium using the Generalized Born model. A set of three different internal dielectric constants, to mimic the different degree of relaxation of the interface when different types of amino acids are mutated for alanine, have to be used for the proteins, depending on the type of amino acid that is mutated. This method permits a systematic scanning mutagenesis of protein-protein interfaces and it is capable of anticipating the experimental results of mutagenesis, thus guiding new experimental investigations.

Caenorhabditis elegans nematodes produce and maintain imprints of attractive chemosensory cues to which they are exposed early in life. Early odor-exposure increases adult chemo-attraction to the same cues. Imprinting is transiently or stably inherited, depending on the number of exposed generations. We show here that the Alanine tRNA (UGC) plays a central role in regulating C. elegans chemo-attraction. Naive worms fed on tRNAAla (UGC) purified from odor-experienced worms, acquire odor-specific imprints. Chemo-attractive responses require the tRNA-modifying Elongator complex sub-units 1 (elpc-1) and 3 (elpc-3) genes. elpc-3 deletions impair chemo-attraction, which is fully restored by wild-type tRNAAla (UGC) feeding. A stably inherited decrease of odor-specific responses ensues from early odor-exposition of elpc-1 deletion mutants. tRNAAla (UGC) may adopt various chemical forms to mediate the cross-talk between innately-programmed and environment-directed chemo-attractive behavior.

Non-small-cell lung cancer (NSCLC) cell lines vary in their sensitivity to glutaminase inhibitors, so it is important to identify the metabolic assets underling their efficacy in cancer cells. Even though specific genetic lesions such as in KRAS and LKB1 have been associated with reliance on glutamine for their metabolic needs, we found no distinction between glutaminase inhibitor CB-839 sensitivity and resistant phenotypes in NSCLC cells with or without these genetic alterations. We demonstrated the close relationship between environmental alanine uptake and catabolism. This response depended on the individual cell's ability to employ alanine aminotransferase (GPT2) to compensate the reduced glutamate availability. It may, therefore, be useful to determine GPT2 levels to predict which NSCLC patients would benefit most from glutaminase inhibitor treatment.

β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9Mab-24 (rat IgG2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1-19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A-F14A, F14A-D15A, D16A-F17A, and F17A-S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target-antibody interaction.

Trypanosoma brucei is a species of unicellular parasite that can cause severe diseases in livestock and humans, including African trypanosomiasis and Chagas disease. Adaptation to diverse environments and changes in nutritional conditions is essential for T. brucei to establish an infection when changing hosts or during invasion of different host tissues. One such adaptation is the ability of T. brucei to rapidly switch its energy metabolism from glucose metabolism in the mammalian blood to proline catabolism in the insect stages and vice versa. However, the mechanisms that support the parasite's response to nutrient availability remain unclear. Using RNAseq and qRT-PCR, we investigated the response of T. brucei to amino acid or glucose starvation and found increased mRNA levels of several amino acid transporters, including all genes of the amino acid transporter AAT7-B subgroup. Functional characterization revealed that AAT7-B members are plasma membrane-localized in T. brucei and when expressed in Saccharomyces cerevisiae supported the uptake of proline, alanine, and cysteine, while other amino acids were poorly recognized. All AAT7-B members showed a preference for proline, which is transported with high or low affinity. RNAi-mediated AAT7-B downregulation resulted in a reduction of intracellular proline concentrations and growth arrest under low proline availability in cultured procyclic form parasites. Taken together, these results suggest a role of AAT7-B transporters in the response of T. brucei to proline starvation and proline catabolism.