Enzymic studies on the biosynthesis of amino acids from lactate by peptostreptococcus elsdenii.
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ABSTRACT:
SUBMITTER: Somerville HJ
PROVIDER: S-EPMC1198775 | biostudies-other | 1968 Jun
REPOSITORIES: biostudies-other
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Project description:Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-(14)C]- or [2-(14)C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of (14)C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C(3) unit derived directly from lactate with a C(1) unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA-->crotonyl-CoA-->glutaconate-->glutamate.
| S-EPMC1198301 | biostudies-other
Project description:1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate (14)C from [1-(14)C]isobutyrate into the valine of cell protein. With P. elsdenii some of the (14)C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate (14)C from [1-(14)C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate (14)C from NaH(14)CO(3) into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, (14)C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, (14)C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system (14)C from [1-(14)C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate-->isobutyryl-CoA-->2-oxoisovalerate-->valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.
| S-EPMC1176590 | biostudies-other
Project description:How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.
| S-EPMC10810186 | biostudies-literature
Project description:Background: Megasphaera elsdenii is an ecologically important rumen bacterium that metabolizes lactate and relieves rumen acidosis (RA) induced by a high-grain-diet. Understanding the regulatory mechanisms of the lactate metabolism of this species in RA conditions might contribute to developing dietary strategies to alleviate RA. Methods: Megasphaera elsdenii was co-cultured with four lactate producers (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, and Selenomonas ruminantium) and a series of substrate starch doses (1, 3, and 9 g/L) were used to induce one normal and two RA models (subacute rumen acidosis, SARA and acute rumen acidosis, ARA) under batch conditions. The associations between bacterial competition and the shift of organic acids' (OA) accumulation patterns in both statics and dynamics manners were investigated in RA models. Furthermore, we examined the effects of substrate lactate concentration and pH on Megasphaera elsdenii's lactate degradation pattern and genes related to the lactate utilizing pathways in the continuous culture. Results and Conclusion: The positive growth of M. elsdenii and B. fibrisolvens caused OA accumulation in the SARA model to shift from lactate to butyrate and resulted in pH recovery. Furthermore, both the quantities of substrate lactate and pH had remarkable effects on M. elsdenii lactate utilization due to the transcriptional regulation of metabolic genes, and the lactate utilization in M. elsdenii was more sensitive to pH changes than to the substrate lactate level. In addition, compared with associations based on statics data, associations discovered from dynamics data showed greater significance and gave additional explanations regarding the relationships between bacterial competition and OA accumulation.
| S-EPMC6374331 | biostudies-literature
Project description:Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.
| S-EPMC4450436 | biostudies-literature
Project description:Butyryl-CoA dehydrogenase prepared by a simple procedure from Peptostreptococcus elsdenii has a molecular weight of approx. 150000. The enzyme has FAD as its prosthetic group. The amino acid analysis is reported. This enzyme, like most of the corresponding mammalian ones, is green. The absorption band at 710nm can be abolished irreversibly by dithionite reduction and air reoxidation; it can be abolished reversibly by phenylmercuric acetate or potassium bromide. The enzyme as isolated appears to be a mixture of a green and a yellow form, both of which are active. This view is supported by the variable ;greenness' of different preparations and the biphasic curve obtained in anaerobic spectrophotometric titrations with dithionite. It can be calculated from the titration results that fully green enzyme would have a peak-to-peak absorption ratio (E(710)/E(430)) as great as 0.54. The green form is much less rapidly reduced by dithionite than the yellow form, but is nevertheless much more readily reduced by dithionite than the enzyme from pig liver. It is also more readily reoxidized by air and shows less tendency to form a semiquinone. Treatment with sodium borohydride produces an unusual reduced species that is probably the 3,4-dihydroflavin.
| S-EPMC1178194 | biostudies-other
Project description:The synthesis of six nonproteinogenic amino acids appropriately protected for Fmoc-based solid-phase peptide synthesis is described. These amino acids are (2S,3R)-vinylthreonine, (2S)-(E)-2-amino-5-fluoro-pent-3-enoic acid (fluoroallylglycine), (S)-beta(2)-homoserine, (S) and (R)-beta(3)-homocysteine, and (2R,3R)-methylcysteine. Once incorporated into peptides, these compounds were envisioned to serve as alternative substrates for the lantibiotic synthases that dehydrate serine and threonine residues in their peptide substrates and catalyze the subsequent intramolecular Michael-type addition of cysteines to the dehydroamino acids.
| S-EPMC2525736 | biostudies-literature
Project description:A subset of natural products, such as polyketides and nonribosomal peptides, is biosynthesized while tethered to a carrier peptide via a thioester linkage. Recently, we reported that the biosyntheses of 3-thiaglutamate and ammosamide, single amino acid-derived natural products, employ a very different type of carrier peptide to which the biosynthetic intermediates are bound via an amide linkage. During their biosyntheses, a peptide aminoacyl-transfer ribonucleic acid (tRNA) ligase (PEARL) first loads an amino acid to the C terminus of the carrier peptide for subsequent modification by other enzymes. Proteolytic removal of the modified C-terminal amino acid yields the mature product. We termed natural products that are biosynthesized using such pathways pearlins. To investigate the diversity of pearlins, in this study we experimentally characterized another PEARL-encoding biosynthetic gene cluster (BGC) from Tistrella mobilis (tmo). The enzymes encoded in the tmo BGC transformed cysteine into 3-thiahomoleucine both in vitro and in Escherichia coli. During this process, a cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme catalyzes C-isopropylation. This work illustrates that the biosynthesis of amino acid-derived natural products on a carrier peptide is a widespread strategy in nature and expands the spectrum of thiahemiaminal analogs of amino acids that may serve a broader, currently unknown function.
| S-EPMC9303977 | biostudies-literature
Project description:BackgroundUpregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined.MethodsWe explore human triple negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received IV infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes.FindingsTNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceeds that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer.ConclusionsGlucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the TCA cycle locally within human breast tumors.
| S-EPMC8248508 | biostudies-literature
Project description:1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.
| S-EPMC1178962 | biostudies-other