Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

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The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

Biochemical Journal ◽

10.1042/bj1220267 ◽

1971 ◽

Vol 122 (3) ◽

pp. 267-276 ◽

Cited By ~ 10

Author(s):

D. C. N. Earl ◽

Susan T. Hindley

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Donor Site ◽

Peptide Bond ◽

Rate Limiting Step ◽

Limiting Step ◽

Donor And Acceptor ◽

Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

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The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

Applied and Environmental Microbiology ◽

10.1128/aem.64.6.1991-1996.1998 ◽

1998 ◽

Vol 64 (6) ◽

pp. 1991-1996 ◽

Cited By ~ 11

Author(s):

Benedicte Flambard ◽

Sandra Helinck ◽

Jean Richard ◽

Vincent Juillard

Keyword(s):

Amino Acids ◽

Growth Rate ◽

Amino Acid ◽

Lactococcus Lactis ◽

Cell Envelope ◽

Essential Amino Acids ◽

Genetically Engineered ◽

Amino Acid Requirements ◽

Rate Limiting

ABSTRACT The ability of caseins to fulfill the amino acid requirements ofLactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

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Methionine coordinates a hierarchically organized anabolic program enabling proliferation

10.1101/249367 ◽

2018 ◽

Cited By ~ 2

Author(s):

Adhish S. Walvekar ◽

Rajalakshmi Srinivasan ◽

Ritu Gupta ◽

Sunil Laxman

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Metabolic Flux ◽

Response Regulator ◽

Pentose Phosphate ◽

Dependent Manner ◽

Comparative Transcriptome ◽

Starvation Stress ◽

Underlying Basis ◽

Rate Limiting

AbstractMethionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here, we construct the organization of this methionine mediated anabolic program, using yeast. Combining comparative transcriptome analysis, biochemical and metabolic flux based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of three key regulatory nodes. These are: the pentose phosphate pathway coupled with reductive biosynthesis, and overall transamination capacity, including the synthesis of glutamate/glutamine. These provides the cofactors or substrates that enhance subsequent rate-limiting reactions in the synthesis of costly amino acids, and nucleotides, which are also induced in a methionine dependent manner. This thereby results in a biochemical cascade establishing an overall anabolic program. For this methionine mediated anabolic program leading to proliferation, cells co-opt a “starvation stress response” regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

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PROGESTERONE AND INSULIN-RESISTANCE: STUDIES OF PROGESTERONE ACTION ON GLUCOSE TRANSPORT, LIPOGENESIS AND LIPOLYSIS IN ISOLATED FAT CELLS OF THE FEMALE RAT

Journal of Endocrinology ◽

10.1677/joe.0.0880455 ◽

1981 ◽

Vol 88 (3) ◽

pp. 455-462 ◽

Cited By ~ 22

Author(s):

M.-TH. SUTTER-DUB ◽

B. DAZEY ◽

E. HAMDAN ◽

M.-TH. VERGNAUD

Keyword(s):

Glucose Metabolism ◽

Pentose Phosphate ◽

Fat Cells ◽

Female Rat ◽

Fat Cell ◽

Rate Limiting Step ◽

Limiting Step ◽

Glucose Phosphorylation ◽

Rate Limiting

The effects of progesterone on isolated rat adipocytes were studied in vitro during various steps of glucose metabolism, transport, lipogenesis and lipolysis. Progesterone decreased the phosphorylation of glucose into glucose-6-phosphate as assessed by measuring the uptake of 2-deoxyglucose but it had no effect on transmembrane transport of glucose as determined by measuring the entry of 3-0-methylglucose into the cell. As glucose phosphorylation is a rate-limiting step of the pentose-phosphate pathway, these data could explain the inhibition of lipogenesis and the enhancement of lipolysis observed when progesterone is present in incubation medium. Progesterone might thus modulate a regulatory step of glucose metabolism and antagonize insulin action in the fat cell.

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GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae

10.1101/819508 ◽

2019 ◽

Author(s):

Khushboo Borah ◽

Jacque-Lucca Kearney ◽

Ruma Banerjee ◽

Pankaj Vats ◽

Huihai Wu ◽

...

Keyword(s):

Amino Acids ◽

Metabolic Network ◽

In Silico ◽

Metabolic Pathways ◽

Mycobacterium Leprae ◽

Human Pathogen ◽

Intracellular Growth ◽

A Genome ◽

Genome Scale

AbstractLeprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen’s obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis (DPA) to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.Author SummaryMycobacterium leprae, the obligate human pathogen is uncultivable in axenic growth medium, and this hinders research on this pathogen, and the pathogenesis of leprosy. The development of novel therapeutics relies on the understanding of growth, survival and metabolism of this bacterium in the host, the knowledge of which is currently very limited. Here we reconstructed a metabolic network of M. leprae-GSMN-ML, a powerful in silico tool to study growth and metabolism of the leprosy bacillus. We demonstrate the application of GSMN-ML to identify the metabolic pathways, and metabolite classes that M. leprae utilizes during intracellular growth.

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The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers

Biochemical Journal ◽

10.1042/bj3450129 ◽

1999 ◽

Vol 345 (1) ◽

pp. 129-138 ◽

Cited By ~ 17

Author(s):

Magali PARIAT ◽

Catherine SALVAT ◽

Magali BÉBIEN ◽

Frédérique BROCKLY ◽

Eléna ALTIERI ◽

...

Keyword(s):

Amino Acids ◽

Structural Integrity ◽

Quaternary Structure ◽

Cysteine Proteases ◽

Site Directed Mutagenesis ◽

Fos Protein ◽

Highly Sensitive ◽

Strict Specificity ◽

Rate Limiting

Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to Pʹ2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.

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Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate

Biochemical Journal ◽

10.1042/bj2230113 ◽

1984 ◽

Vol 223 (1) ◽

pp. 113-117 ◽

Cited By ~ 7

Author(s):

T N Palmer ◽

M A Caldecourt ◽

M C Sugden

Keyword(s):

Amino Acids ◽

Pyruvate Dehydrogenase ◽

Tricarboxylic Acid Cycle ◽

Acid Oxidation ◽

14Co2 Production ◽

In Vitro Effects ◽

Tricarboxylic Acid Cycle Intermediates ◽

Rate Limiting ◽

Main Carbon

Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle.

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A signal-anchor sequence selective for the mitochondrial outer membrane.

The Journal of Cell Biology ◽

10.1083/jcb.119.6.1451 ◽

1992 ◽

Vol 119 (6) ◽

pp. 1451-1457 ◽

Cited By ~ 83

Author(s):

H M McBride ◽

D G Millar ◽

J M Li ◽

G C Shore

Keyword(s):

Amino Acids ◽

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Hybrid Protein ◽

Transmembrane Segment ◽

Structural Domains ◽

Signal Anchor ◽

Anchor Sequence ◽

Rate Limiting

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

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A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650145 ◽

1981 ◽

Vol 45 (02) ◽

pp. 110-115 ◽

Cited By ~ 5

Author(s):

György Csákó ◽

Eva A Suba

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Platelet Reactivity ◽

High Specificity ◽

Turbidimetric Method ◽

Specific Manner ◽

Aggregation Inhibition ◽

Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

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Synthesis and Breakdown of the Universal Precursors to Biological Metabolism Promoted by Ferrous Iron

10.26434/chemrxiv.7178930.v1 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kamila B. Muchowska ◽

Sreejith Jayasree VARMA ◽

Joseph Moran

Keyword(s):

Amino Acids ◽

Chemical Reaction ◽

Metabolic Pathways ◽

Ferrous Iron ◽

Reaction Network ◽

Tca Cycle ◽

Chemical Reaction Network ◽

Metabolic Precursors ◽

Tca Cycle Intermediates

How core biological metabolism initiated and why it uses the intermediates, reactions and pathways that it does remains unclear. Life builds its molecules from CO2 and breaks them down to CO2 again through the intermediacy of just five metabolites that act as the hubs of biochemistry. Here, we describe a purely chemical reaction network promoted by Fe2+ in which aqueous pyruvate and glyoxylate, two products of abiotic CO2 reduction, build up nine of the eleven TCA cycle intermediates, including all five universal metabolic precursors. The intermediates simultaneously break down to CO2 in a life-like regime resembling biological anabolism and catabolism. Introduction of hydroxylamine and Fe0 produces four biological amino acids. The network significantly overlaps the TCA/rTCA and glyoxylate cycles and may represent a prebiotic precursor to these core metabolic pathways.

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