Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

PubMed

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

2014-11-25

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

PubMed Central

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

2014-01-01

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

PubMed Central

Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

2015-01-01

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

Lipoic acid synthetase deficiency causes neonatal-onset epilepsy, defective mitochondrial energy metabolism, and glycine elevation.

PubMed

Mayr, Johannes A; Zimmermann, Franz A; Fauth, Christine; Bergheim, Christa; Meierhofer, David; Radmayr, Doris; Zschocke, Johannes; Koch, Johannes; Sperl, Wolfgang

2011-12-09

Lipoic acid is an essential prosthetic group of four mitochondrial enzymes involved in the oxidative decarboxylation of pyruvate, α-ketoglutarate, and branched chain amino acids and in the glycine cleavage. Lipoic acid is synthesized stepwise within mitochondria through a process that includes lipoic acid synthetase. We identified the homozygous mutation c.746G>A (p.Arg249His) in LIAS in an individual with neonatal-onset epilepsy, muscular hypotonia, lactic acidosis, and elevated glycine concentration in plasma and urine. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate and decreased pyruvate dehydrogenase complex activity. A pronounced reduction of the prosthetic group lipoamide was found in lipoylated proteins. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Peptide Synthetase Gene in Trichoderma virens

PubMed Central

Wilhite, S. E.; Lumsden, R. D.; Straney, D. C.

2001-01-01

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated Nδ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used. PMID:11679326

The Aminoacyl-tRNA Synthetase Complex.

PubMed

Mirande, Marc

2017-01-01

Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.

A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

PubMed

Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

2005-04-01

Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.

PubMed

Armen, Roger S; Schiller, Stefan M; Brooks, Charles L

2010-06-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

PubMed Central

Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

2015-01-01

Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

PubMed

Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-06-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

PubMed

Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

1976-07-01

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

Steric and Thermodynamic Limits of Design for the Incorporation of Large UnNatural Amino Acids in Aminoacyl-tRNA Synthetase Enzymes

PubMed Central

Armen, Roger S.; Schiller, Stefan M.; Brooks, Charles L.

2015-01-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-α-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. PMID:20310065

Glutamate-Dependent Translational Control of Glutamine Synthetase in Bergmann Glia Cells.

PubMed

Tiburcio-Félix, Reynaldo; Escalante-López, Miguel; López-Bayghen, Bruno; Martínez, Daniel; Hernández-Kelly, Luisa C; Zinker, Samuel; Hernández-Melchor, Dinorah; López-Bayghen, Esther; Olivares-Bañuelos, Tatiana N; Ortega, Arturo

2018-06-01

Glutamate is the major excitatory transmitter of the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed both in neurons and in glial cells. Recent evidence has shown that glutamate uptake systems, particularly enriched in glia cells, trigger biochemical cascades in a similar fashion as receptors. A tight regulation of glutamate extracellular levels prevents neuronal overstimulation and cell death, and it is critically involved in glutamate turnover. Glial glutamate transporters are responsible of the majority of the brain glutamate uptake activity. Once internalized, this excitatory amino acid is rapidly metabolized to glutamine via the astrocyte-enriched enzyme glutamine synthetase. A coupling between glutamate uptake and glutamine synthesis and release has been commonly known as the glutamate/glutamine shuttle. Taking advantage of the established model of cultured Bergmann glia cells, in this contribution, we explored the gene expression regulation of glutamine synthetase. A time- and dose-dependent regulation of glutamine synthetase protein and activity levels was found. Moreover, glutamate exposure resulted in the transient shift of glutamine synthetase mRNA from the monosomal to the polysomal fraction. These results demonstrate a novel mode of glutamate-dependent glutamine synthetase regulation and strengthen the notion of an exquisite glia neuronal interaction in glutamatergic synapses.

Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling

PubMed Central

Zhu, Jianzhong; Zhang, Yugen; Ghosh, Arundhati; Cuevas, Rolando A.; Forero, Adriana; Dhar, Jayeeta; Ibsen, Mikkel Søes; Schmid-Burgk, Jonathan Leo; Schmidt, Tobias; Ganapathiraju, Madhavi K.; Fujita, Takashi; Hartmann, Rune; Barik, Sailen; Hornung, Veit; Coyne, Carolyn B.; Sarkar, Saumendra N.

2014-01-01

SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation. PMID:24931123

Pseudouridylate Synthetase of Escherichia coli: a Catabolite-Repressible Enzyme

PubMed Central

Solomon, L. R.; Breitman, T. R.

1971-01-01

The growth on pseudouridine of two pyrimidine auxotrophs of Escherichia coli (Bu− and W63-86) was markedly enhanced when glycerol replaced glucose as a carbon source or when adenosine 3′:5′-cyclic monophosphoric acid was added to medium containing glucose. These results indicated that an enzyme catalyzing a reaction in the pathway of pseudouridine conversion to uracil was sensitive to catabolite repression. The following pathway is proposed for pseudouridine utilization: [Formula: see text] [Formula: see text] Pseudouridylate synthetase was sensitive to catabolite repression in strains Bu− and W63-86. In contrast, strains B5RU and W5RU, mutants of Bu− and W63-86 which were selected for their ability to grow rapidly on pseudouridine in the presence of glucose, had high levels of pseudouridylate synthetase in the presence of glucose. In the case of B5RU but not W5RU, synthetase activity was greater in cells grown on glycerol or on glucose plus adenosine 3′:5-cyclic monophosphoric acid than on glucose. PMID:4329733

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

PubMed Central

Clemente, Maria R.; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K.; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-01-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses. PMID:22442424

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis.

PubMed

Braga, Daniel; Hoffmeister, Dirk; Nett, Markus

2016-01-01

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

DOE PAGES

Crnkovic, Ana; Suzuki, Tateki; Soll, Dieter; ...

2016-06-14

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encodedmore » amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.« less

Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

NASA Technical Reports Server (NTRS)

Max, Stephen R.; Konagaya, Masaaki; Konagaya, Yoko; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa

1986-01-01

The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

PubMed

Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

2013-04-02

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

PubMed

Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

2015-01-01

Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

PubMed Central

Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

2013-01-01

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

Transfer of D-phenylalanine from gramicidin S synthetase 1 to gramicidin S synthetase 2 in gramicidin S synthesis.

PubMed

Hori, K; Kanda, M; Miura, S; Yamada, Y; Saito, Y

1983-01-01

The transfer of phenylalanine from gramicidin S synthetase 1 (GS 1) to gramicidin S synthetase 2 (GS 2) was studied by the use of combinations of wild-type GS 1 with various GS 2s from a wild strain and gramicidin S non-producing mutant strains of Bacillus brevis Nagano. The combinations of mutant GS 2s lacking 4'-phosphopantetheine (from BI-4, C-3, E-1, and E-2) did not transfer D-phenylalanine from GS 1, although they could activate all the constituent amino acids. Other mutant GS 2s containing 4'-phosphopantetheine, except GS 2 from BII-3 (proline-activation lacking) accepted D-phenylalanine from intact GS 1. To ascertain more directly whether 4'-phosphopantetheine is involved in the transfer of D-phenylalanine from GS 1 to GS 2, pepsin digests of GS 2 that accepted [14C]phenylalanine were analyzed by Sephadex G-50 column chromatography and thin-layer chromatography (TLC). Radioactivity of [14C]phenylalanine was always associated with a peptide containing 4'-phosphopantetheine. Furthermore, the position of radioactivity was distinct from the position of 4'-phosphopantetheine on TLC after alkaline treatment or performic acid oxidation of the digests.

Rational design of aminoacyl-tRNA synthetase specific for p-acetyl-L-phenylalanine.

PubMed

Sun, Renhua; Zheng, Heng; Fang, Zhengzhi; Yao, Wenbing

2010-01-01

The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations. Copyright 2009 Elsevier Inc. All rights reserved.

Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

PubMed

Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

2016-11-10

Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

Versatility of acyl-acyl carrier protein synthetases

DOE PAGES

Beld, Joris; Finzel, Kara; Burkart, Michael D.

2014-10-09

The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

PubMed

Stephen, Preyesh; Ye, Sheng; Zhou, Ming; Song, Jian; Zhang, Rongguang; Wang, En-Duo; Giegé, Richard; Lin, Sheng-Xiang

2018-05-25

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNA Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of GSH1 and GSH2 Gene Mutation on Glutathione Synthetases Activity of Saccharomyces cerevisiae.

PubMed

Xu, Wen; Jia, Haiyan; Zhang, Longmei; Wang, Haiyan; Tang, Hui; Zhang, Liping

2017-08-01

In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg 2+ /ADP complex in the overall GCL structure. L51P mutant spread distortion on the β-sheet due to the fact that the φ was changed from -50.4° to -40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a β-sheet. The results confirm that introduction of proline located at the middle of the β-sheet or at the N- or C-terminal between α-helix and β-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.

A Survey of Glutamine Synthetase Activities in Tissues from Three Classes of Fish.

DTIC Science & Technology

1980-09-01

reveree side it necessay end identify by block enamaber) Glutamine synthetase, gamma-glutamyl transferase, osmoregulation , glutamate, glutamine...aspects of osmoregulation as well. The only known route of glutanmine synthesis n all species is activity of glutamine synthetase (EC 6.3.1.2) which...for osmoregulation . There is a relatively small difference n species which retain urea for osmoregulation . This may help to explain the relationship of

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolutionmore » crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.« less

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

PubMed Central

Mérida, A; Candau, P; Florencio, F J

1991-01-01

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium. Images PMID:1676397

Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase.

PubMed

Iqbal, Emil S; Dods, Kara K; Hartman, Matthew C T

2018-02-14

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic β-amino acids.

An acyl-CoA synthetase in Mycobacterium tuberculosis involved in triacylglycerol accumulation during dormancy.

PubMed

Daniel, Jaiyanth; Sirakova, Tatiana; Kolattukudy, Pappachan

2014-01-01

Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids.

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

PubMed Central

Heyes, Derren J.; Levy, Colin; Lafite, Pierre; Roberts, Ian S.; Goldrick, Marie; Stachulski, Andrew V.; Rossington, Steven B.; Stanford, Deborah; Rigby, Stephen E. J.; Scrutton, Nigel S.; Leys, David

2009-01-01

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg2+ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp100 and Asp235 in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group. PMID:19815542

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

PubMed

Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

2015-04-01

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

PubMed Central

Neuberger, Albert; Sandy, John D.; Tait, George H.

1973-01-01

1. The `initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the `maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80–90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65–75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the `low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the `high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH

The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, S.M.; Habash, D.Z.

2009-07-02

Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulationmore » of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.« less

Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

PubMed Central

Jiang, D W; Englund, P T

2001-01-01

As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

PubMed Central

Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

2014-01-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

PubMed

Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

2013-11-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

A high-throughput screen against pantothenate synthetase (PanC) identifies 3-biphenyl-4-cyanopyrrole-2-carboxylic acids as a new class of inhibitor with activity against Mycobacterium tuberculosis.

PubMed

Kumar, Anuradha; Casey, Allen; Odingo, Joshua; Kesicki, Edward A; Abrahams, Garth; Vieth, Michal; Masquelin, Thierry; Mizrahi, Valerie; Hipskind, Philip A; Sherman, David R; Parish, Tanya

2013-01-01

The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

PubMed Central

Anderson, P M

1989-01-01

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for

Acetate scavenging activity in Escherichia coli: interplay of acetyl-CoA synthetase and the PEP-glyoxylate cycle in chemostat cultures.

PubMed

Renilla, Sergio; Bernal, Vicente; Fuhrer, Tobias; Castaño-Cerezo, Sara; Pastor, José M; Iborra, José L; Sauer, Uwe; Cánovas, Manuel

2012-03-01

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl-CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl-CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl-CoA synthetase are upregulated. A mutant in acs (encoding acetyl-CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl-CoA synthetase by cAMP-CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl-CoA and acetyl-phosphate pools, although establishing an energy-dissipating substrate cycle.

Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

PubMed

Sharma, Arvind; Sharma, Amit

2015-02-01

The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize.

PubMed

Prinsi, Bhakti; Espen, Luca

2015-04-03

Glutamine synthetase (GS) catalyzes the first step of nitrogen assimilation in plant cell. The main GS are classified as cytosolic GS1 and plastidial GS2, of which the functionality is variable according to the nitrogen sources, organs and developmental stages. In maize (Zea mays L.) one gene for GS2 and five genes for GS1 subunits are known, but their roles in root metabolism are not yet well defined. In this work, proteomic and biochemical approaches have been used to study root GS enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. The plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. The analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six GS proteins. Three forms of GS2 were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial GS activity. Nitrogen availabilities enabled increments in root total GS synthetase activity, associated with different GS1 isoforms according to the nitrogen sources. Nitrate nutrition induced the specific accumulation of GS1-5 while ammonium led to up-accumulation of both GS1-1 and GS1-5, highlighting co-participation. Moreover, the changes in thermal sensitivity of root GS transferase activity suggested differential rearrangements of the native enzyme. The amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. Glutamine showed the prevalent changes in all nitrogen nutritions. Besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate. This work provides new information about the multifaceted regulation of the GS enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post

Crystal structure of an indole-3-acetic acid amido synthetase from grapevine involved in auxin homeostasis.

PubMed

Peat, Thomas S; Böttcher, Christine; Newman, Janet; Lucent, Del; Cowieson, Nathan; Davies, Christopher

2012-11-01

Auxins are important for plant growth and development, including the control of fruit ripening. Conjugation to amino acids by indole-3-acetic acid (IAA)-amido synthetases is an important part of auxin homeostasis. The structure of the auxin-conjugating Gretchen Hagen3-1 (GH3-1) enzyme from grapevine (Vitis vinifera), in complex with an inhibitor (adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate), is presented. Comparison with a previously published benzoate-conjugating enzyme from Arabidopsis thaliana indicates that grapevine GH3-1 has a highly similar domain structure and also undergoes a large conformational change during catalysis. Mutational analyses and structural comparisons with other proteins have identified residues likely to be involved in acyl group, amino acid, and ATP substrate binding. Vv GH3-1 is a monomer in solution and requires magnesium ions solely for the adenlyation reaction. Modeling of IAA and two synthetic auxins, benzothiazole-2-oxyacetic acid (BTOA) and 1-naphthaleneacetic acid (NAA), into the active site indicates that NAA and BTOA are likely to be poor substrates for this enzyme, confirming previous enzyme kinetic studies. This suggests a reason for the increased effectiveness of NAA and BTOA as auxins in planta and provides a tool for designing new and effective auxins.

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa

Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligandmore » complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.« less

Functional expansion of human tRNA synthetases achieved by structural inventions

PubMed Central

Guo, Min; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions. PMID:19932696

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melton, Elaina M.; Center for Cardiovascular Sciences, Albany Medical College, Albany, NY; Cerny, Ronald L.

Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4,more » for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

NASA Technical Reports Server (NTRS)

Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

1987-01-01

The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

Role of coupled dynamics in the catalytic activity of prokaryotic-like prolyl-tRNA synthetases.

PubMed

Sanford, Brianne; Cao, Bach; Johnson, James M; Zimmerman, Kurt; Strom, Alexander M; Mueller, Robyn M; Bhattacharyya, Sudeep; Musier-Forsyth, Karin; Hati, Sanchita

2012-03-13

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

NASA Astrophysics Data System (ADS)

Aboelnga, Mohamed M.

cysteine and valine, respectively. In chapter 8, an assessment QM/MM study using a variety of DFT functionals to represent the chemically active layer in aminoacylation mechanism of the unnatural amino acid ss-Hydroxynorvaline as catalyzed by Threonyl-tRNA synthetase has been carried out. Overall, it was found that substrate-assisted mechanisms are a common pathway for these enzymes. One important application of such information is to establish the criteria required for any candidate to inhibit the catalytic functions of aaRS, which was applied in chapter 9 to screen potential competitive inhibitors able to efficiently block the bacterial Threonyl-tRNA synthetases. The investigations reported herein should provide atomistic details into the fundamental catalytic mechanisms of the ubiquitous and ancient aaRS enzymes. Consequently, they will also help enable a much-needed deeper understanding of the underlying chemical principles of catalysis in general.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

PubMed Central

Weigel, B J; Burgett, S G; Chen, V J; Skatrud, P L; Frolik, C A; Queener, S W; Ingolia, T D

1988-01-01

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution. Images PMID:3045077

Identification, Expression and IAA-Amide Synthetase Activity Analysis of Gretchen Hagen 3 in Papaya Fruit (Carica papaya L.) during Postharvest Process

PubMed Central

Liu, Kaidong; Wang, Jinxiang; Li, Haili; Zhong, Jundi; Feng, Shaoxian; Pan, Yaoliang; Yuan, Changchun

2016-01-01

Auxin plays essential roles in plant development. Gretchen Hagen 3 (GH3) genes belong to a major auxin response gene family and GH3 proteins conjugate a range of acylsubstrates to alter the levels of hormones. Currently, the role of GH3 genes in postharvest physiological regulation of ripening and softening processes in papaya fruit is unclear. In this study, we identified seven CpGH3 genes in a papaya genome database. The CpGH3.1a, CpGH3.1b, CpGH3.5, CpGH3.6, and CpGH3.9 proteins were identified as indole-3-acetic acid (IAA)-specific amido synthetases. We analyzed the changes in IAA-amido synthetase activity using aspartate as a substrate for conjugation and found a large increase (over 5-fold) during the postharvest stages. Ascorbic acid (AsA) application can extend the shelf life of papaya fruit. Our data showed that AsA treatment regulates postharvest fruit maturation processes by promoting endogenous IAA levels. Our findings demonstrate the important role of GH3 genes in the regulation of auxin-associated postharvest physiology in papaya. PMID:27812360

Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

PubMed

Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

2016-01-01

Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens)

Treesearch

Michelle J. Serapiglia; Rakesh Minocha; Subhash C. Minocha

2008-01-01

We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount...

Function of membranous lysyl-tRNA synthetase and its implication for tumorigenesis.

PubMed

Young, Ho Jeon; Lee, Jung Weon; Kim, Sunghoon

2016-12-01

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that conjugate specific amino acids to their cognate tRNAs for protein synthesis. Besides their catalytic activity, recent studies have uncovered many additional functions of these enzymes through their interactions with diverse cellular factors. Among human ARSs, cytosolic lysyl-tRNA synthetase (KRS) is often highly expressed in cancer cells and tissues, and facilitates cancer cell migration and invasion through the interaction with the 67kDa laminin receptor on the plasma membrane. Specific modulation of this interaction by small molecule inhibitors has revealed a new way to control metastasis. Here, we summarize the pro-metastatic functions of KRS and their patho-physiological implications. Copyright © 2016 Elsevier B.V. All rights reserved.

Selective and Specific Inhibition of the Plasmodium falciparum Lysyl-tRNA Synthetase by the Fungal Secondary Metabolite Cladosporin

PubMed Central

Hoepfner, Dominic; McNamara, Case W.; Lim, Chek Shik; Studer, Christian; Riedl, Ralph; Aust, Thomas; McCormack, Susan L.; Plouffe, David M.; Meister, Stephan; Schuierer, Sven; Plikat, Uwe; Hartmann, Nicole; Staedtler, Frank; Cotesta, Simona; Schmitt, Esther K.; Petersen, Frank; Supek, Frantisek; Glynne, Richard J.; Tallarico, John A.; Porter, Jeffrey A.; Fishman, Mark C.; Bodenreider, Christophe; Diagana, Thierry T.; Movva, N. Rao; Winzeler, Elizabeth A.

2012-01-01

Summary With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited. PMID:22704625

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

PubMed

Hoepfner, Dominic; McNamara, Case W; Lim, Chek Shik; Studer, Christian; Riedl, Ralph; Aust, Thomas; McCormack, Susan L; Plouffe, David M; Meister, Stephan; Schuierer, Sven; Plikat, Uwe; Hartmann, Nicole; Staedtler, Frank; Cotesta, Simona; Schmitt, Esther K; Petersen, Frank; Supek, Frantisek; Glynne, Richard J; Tallarico, John A; Porter, Jeffrey A; Fishman, Mark C; Bodenreider, Christophe; Diagana, Thierry T; Movva, N Rao; Winzeler, Elizabeth A

2012-06-14

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited. Copyright © 2012 Elsevier Inc. All rights reserved.

Arabidopsis thaliana GH3.15 acyl acid amido synthetase has a highly specific substrate preference for the auxin precursor indole-3-butyric acid.

PubMed

Sherp, Ashley M; Westfall, Corey S; Alvarez, Sophie; Jez, Joseph M

2018-03-23

Various phytohormones control plant growth and development and mediate biotic and abiotic stress responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases are plant enzymes that typically conjugate amino acids to indole-3-acetic acid (IAA) or jasmonic acid (JA) to inactivate or activate these phytohormones, respectively; however, the physiological and biological roles of many of these enzymes remain unclear. Using a biochemical approach, we found that the Arabidopsis thaliana GH3.15 (AtGH3.15) preferentially uses indole-3-butyric acid (IBA) and glutamine as substrates. The X-ray crystal structure of the AtGH3.15·AMP complex, modeling of IBA in the active site, and biochemical analysis of site-directed mutants provide insight on active site features that lead to AtGH3.15's preference for IBA. Assay-based in planta analysis of AtGH3.15-overexpressing lines indicated that their root elongation and lateral root density were resistant to IBA treatment but not to treatment with either IAA or JA. These findings suggest that AtGH3.15 may play a role in auxin homeostasis by modulating the levels of IBA for peroxisomal conversion to IAA. Analysis of AtGH3.15 promoter-driven yellow fluorescent protein reporter lines revealed that AtGH3.15 is expressed at significant levels in seedlings, roots, and parts of the siliques. We conclude that AtGH3.15 is unique in the GH3 protein family for its role in modifying IBA in auxin homeostasis and that it is the first GH3 protein shown to primarily modify a plant growth regulator other than IAA and JA. © 2018 Sherp et al.

Role of Coupled-Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases

PubMed Central

Sanford, Brianne; Cao, Bach; Johnson, James M.; Zimmerman, Kurt; Strom, Alexander M.; Mueller, Robyn M.; Bhattacharyya, Sudeep; Musier-Forsyth, Karin; Hati, Sanchita

2012-01-01

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNAPro substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNAPro is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In the present study, experimental and computational approaches were used to test the hypothesis that INS deletion alters the internal protein dynamics leading to reduce catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199–206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled-dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Taken together, the present study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity. PMID:22356126

Structural insights into the polyphyletic origins of glycyl tRNA synthetases

DOE PAGES

Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; ...

2016-05-23

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α 2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α 2β 2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α 2β 2more » GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. Furthermore, a structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α 2β 2 GlyRS, convergent with α 2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.« less

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

PubMed Central

Casabon, Israël; Swain, Kendra; Crowe, Adam M.

2014-01-01

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

Mis-Regulation of 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate Synthetase Does Not Account for Growth Inhibition by Phenylalanine in Agmenellum quadruplicatum

PubMed Central

Jensen, Roy A.; Stenmark-Cox, S.; Ingram, Lonnie O.

1974-01-01

The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis.

PubMed

Lü, Shiyou; Song, Tao; Kosma, Dylan K; Parsons, Eugene P; Rowland, Owen; Jenks, Matthew A

2009-08-01

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

Crystal structure of human cytosolic aspartyl-tRNA synthetase, a component of multi-tRNA synthetase complex

PubMed Central

Kim, Kyung Rok; Park, Sang Ho; Kim, Hyoun Sook; Rhee, Kyung Hee; Kim, Byung-Gyu; Kim, Dae Gyu; Park, Mi Seul; Kim, Hyun-Jung; Kim, Sunghoon; Han, Byung Woo

2013-01-01

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Å2 which comprises 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNAAsp to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions. PMID:23609930

Crystal structure of human cytosolic aspartyl-tRNA synthetase, a component of multi-tRNA synthetase complex.

PubMed

Kim, Kyung Rok; Park, Sang Ho; Kim, Hyoun Sook; Rhee, Kyung Hee; Kim, Byung-Gyu; Kim, Dae Gyu; Park, Mi Seul; Kim, Hyun-Jung; Kim, Sunghoon; Han, Byung Woo

2013-10-01

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Å(2) which comprises 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNA(Asp) to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions. Copyright © 2013 Wiley Periodicals, Inc.

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.

PubMed

Jiang, Yu; Tao, Rongsheng; Shen, Zhengquan; Sun, Liangdong; Zhu, Fuyun; Yang, Sheng

2016-12-01

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l -1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol -1 added cysteine and a productivity of 6.1 ± 0.0 g l -1  h -1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.

Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2) defects predicts differential effects on aminoacylation

PubMed Central

Euro, Liliya; Konovalova, Svetlana; Asin-Cayuela, Jorge; Tulinius, Már; Griffin, Helen; Horvath, Rita; Taylor, Robert W.; Chinnery, Patrick F.; Schara, Ulrike; Thorburn, David R.; Suomalainen, Anu; Chihade, Joseph; Tyynismaa, Henna

2015-01-01

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes. PMID:25705216

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

DOEpatents

Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

2010-05-11

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

2012-05-22

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

2008-04-08

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7T during Degradation of 3,3′-Dithiodipropionic Acid ▿ †

PubMed Central

Schürmann, Marc; Wübbeler, Jan Hendrik; Grote, Jessica; Steinbüchel, Alexander

2011-01-01

The sucCD gene of Advenella mimigardefordensis strain DPN7T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCDAm) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCDAm is Mg2+ or Mn2+ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (Vmax = 9.85 ± 0.14 μmol min−1 mg−1, Km = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (Vmax = 0.12 ± 0.01 μmol min−1 mg−1) and the affinity was 6-fold lower (Km = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCDAm is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP. PMID:21515777

Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

PubMed Central

Hong, Y L; Hossler, P A; Calhoun, D H; Meshnick, S R

1995-01-01

Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia. PMID:7486915

Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases*♦

PubMed Central

Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Brieba, Luis G.; Grøtli, Morten

2016-01-01

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

Adenylosuccinate synthetase: recent developments.

PubMed

Honzatko, R B; Stayton, M M; Fromm, H J

1999-01-01

By exerting strategic control on purine nucleotide biosynthesis, and by engaging GTP-dependent transphosphorylation of IMP to activate loss of an oxygen atom during catalysis, adenylosuccinate synthetase remains as enzyme that justifiably fascinates students of enzyme catalysis. This review describes how the balanced application of X-ray crystallography and enzyme kinetics has advanced the comprehension of the catalytic and regulatory properties of adenylosuccinate synthetase. Detailed analysis has demonstrated the formation of 6-phosphoryl-IMP, an intermediate originally postulated over 40 years ago on the basis of oxygen-18 exchange experiments showing that position-6 oxygen of IMP becomes incorporated into phosphate. Inferences about the participation of amino acid side-chains that stabilize 6-P-IMP during catalysis have also been confirmed by site-directed mutagenesis and examination of such mutations on various kinetic parameters. Moreover, the action of certain regulatory ligands have also been viewed at atomic level resolution. For example, magnesium ion and GDP can induce conformational changes linked to the stabilization of one of two known conformations of the so-called 40s loop. Another significant finding is that two magnesium ions play fundamental roles: one binding with high affinity to the substrate GTP, and a second binding with lower affinity to the co-substrate aspartate. These structural and kinetic studies have also formed the basis for clarifying the action of various inhibitors and potentially important pharmacologic agents with this key regulatory enzyme. Finally, this review explores the current status of investigations on gene structure and gene expression in a number of organisms.

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

PubMed

Fujita, Akiko; Sato, Chihiro; Münster-Kühnel, Anja-K; Gerardy-Schahn, Rita; Kitajima, Ken

2005-02-01

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay

Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wells, P.G.; Zubovits, J.T.; Wong, S.T.

1989-02-01

Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeicmore » acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05).« less

Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan.

PubMed

Liu, Xinyu; Walsh, Christopher T

2009-09-15

The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CpaS, CpaD, and CpaO. CpaS is a hybrid, two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-L-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold. The NRPS module in CpaS has a predicted four-domain organization of condensation, adenylation, thiolation, and reductase* (C-A-T-R*), where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) superfamily. By heterologous overproduction in Escherichia coli of the 56 kDa Aspergillus flavus CpaS TR* didomain and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann-type cyclization on the N-acetoacetyl-Trp intermediate bound in thioester linkage to the phosphopantetheinyl arm of the T domain to form and release cAATrp. This occurs without any participation of NAD(P)H, so R* does not function as a canonical SDR family member. Use of the T and R* domains in in trans assays enabled multiple turnovers and evaluation of specific mutants. Mutation of the D3803 residue in the R* domain, conserved in other fungal tetramate synthetases, abolished activity both in in trans and in cis (TR*) activity assays. It is likely that cyclization of beta-ketoacylaminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines.

TRYPTOPHAN SYNTHETASE LEVELS IN ESCHERICHIA COLI, SHIGELLA DYSENTERIAE, AND TRANSDUCTION HYBRIDS

PubMed Central

Eisenstein, Richard B.; Yanofsky, Charles

1962-01-01

Eisenstein, Richard B. (Western Reserve University, Cleveland, Ohio) and Charles Yanofsky. Tryptophan synthetase levels in Escherichia coli, Shigella dysenteriae, and transduction hybrids. J. Bacteriol. 83:193–204. 1962—Shigella dysenteriae and Escherichia coli, strains K-12 and B, were found to produce low levels of tryptophan synthetase, although some hybrids, formed by the introduction of the gene cluster concerned with tryptophan synthesis from S. dysenteriae into E. coli, produced high levels of this enzyme system. A revertant obtained from a tryptophan-requiring mutant also formed high levels of tryptophan synthetase. The gene or genes responsible for high enzyme production in these strains was shown to be linked to the cluster of genes concerned with tryptophan synthesis. The cause of high enzyme production was investigated. Various lines of evidence, including stimulation of growth by tryptophan precursors, sensitivity to inhibition by 5-methyltryptophan, absence of accumulation of tryptophan, and repression of enzyme formation by anthranilic acid and tryptophan, suggested that high enzyme production in the strains examined results from a partial block in the tryptophan pathway and not from resistance to repression by tryptophan. The conversion of shikimic acid-5-phosphate to anthranilic acid appears to be the partially blocked reaction in the strains studied. PMID:13889700

Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter G.; Wang, Lei; Anderson, John Christopher

2015-10-20

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

DOEpatents

Schultz, Peter; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

2006-08-01

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and composition for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2012-05-08

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and compositions for the production of orthogonal tRNA-aminoacyl-tRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2011-09-06

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.

PubMed

Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D

2008-11-25

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase

Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

USDA-ARS?s Scientific Manuscript database

Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology.

PubMed

Macarrón, R; Mensah, L; Cid, C; Carranza, C; Benson, N; Pope, A J; Díez, E

2000-09-10

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods. Copyright 2000 Academic Press.

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

PubMed Central

Krauss, S. W.; Stollar, B. D.; Friedkin, M.

1973-01-01

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene. Images PMID:4575286

Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation.

PubMed Central

Francklyn, Christopher; Perona, John J; Puetz, Joern; Hou, Ya-Ming

2002-01-01

Aminoacyl-tRNA synthetases attach amino acids to the 3' termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13-18, 2002) discussed new research into the structure-function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code. PMID:12458790

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance.

PubMed

Chen, J.; Goldsbrough, P. B.

1994-09-01

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

PubMed Central

Yocum, R R; Perkins, J B; Howitt, C L; Pero, J

1996-01-01

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli. PMID:8755891

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

PubMed

Yocum, R R; Perkins, J B; Howitt, C L; Pero, J

1996-08-01

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

PubMed

Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A G; Santamaría-Gómez, Javier; Patterson, Carl J; Foster, Andrew W; Bru-Martínez, Roque; Robinson, Nigel J; Luque, Ignacio

2015-11-16

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

PubMed Central

Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

1999-01-01

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant.

PubMed

Exner, Matthias P; Kuenzl, Tilmann; To, Tuyet Mai T; Ouyang, Zhaofei; Schwagerus, Sergej; Hoesl, Michael G; Hackenberger, Christian P R; Lensen, Marga C; Panke, Sven; Budisa, Nediljko

2017-01-03

The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

PubMed

Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

2010-08-10

Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Large-scale filament formation inhibits the activity of CTP synthetase

PubMed Central

Barry, Rachael M; Bitbol, Anne-Florence; Lorestani, Alexander; Charles, Emeric J; Habrian, Chris H; Hansen, Jesse M; Li, Hsin-Jung; Baldwin, Enoch P; Wingreen, Ned S; Kollman, Justin M; Gitai, Zemer

2014-01-01

CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001 PMID:25030911

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

PubMed

Hadd, Andrew; Perona, John J

2014-12-19

We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains.

PubMed

Halawani, Dalia; Gogonea, Valentin; DiDonato, Joseph A; Pipich, Vitaliy; Yao, Peng; China, Arnab; Topbas, Celalettin; Vasu, Kommireddy; Arif, Abul; Hazen, Stanley L; Fox, Paul L

2018-06-08

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH- S -transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

PubMed

Christensen, Karen E; Hou, Wenyang; Bahous, Renata H; Deng, Liyuan; Malysheva, Olga V; Arning, Erland; Bottiglieri, Teodoro; Caudill, Marie A; Jerome-Majewska, Loydie A; Rozen, Rima

2016-11-01

Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. Female Mthfd1S +/+ and Mthfd1S +/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S +/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater

Regulated capture by exosomes of mRNAs for cytoplasmic tRNA synthetases.

PubMed

Wang, Feng; Xu, Zhiwen; Zhou, Jie; Lo, Wing-Sze; Lau, Ching-Fun; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

2013-10-11

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

PubMed Central

Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth

2014-01-01

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. PMID:24562907

Succinyl-CoA Synthetase is a Phosphate Target for the Activation of Mitochondrial Metabolism

PubMed Central

Phillips, Darci; Aponte, Angel M.; French, Stephanie A.; Chess, David J.; Balaban, Robert S.

2009-01-01

Succinyl-CoA synthetase (SCS) is the only mitochondrial enzyme capable of ATP production via substrate level phosphorylation in the absence of oxygen, but it also plays a key role in the citric acid cycle, ketone metabolism and heme synthesis. Inorganic phosphate (Pi) is a signaling molecule capable of activating oxidative phosphorylation at several sites, including NADH generation and as a substrate for ATP formation. In this study it was shown that Pi-binds porcine heart SCS α-subunit (SCSα) in a non-covalent manner and enhances its enzymatic activity, thereby providing a new target for Pi-activation in mitochondria. Coupling 32P-labeling of intact mitochondria with SDS gel electrophoresis revealed that 32P-labeling of SCSα was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS) it was shown that this enhanced 32P-labeling resulted from a simple binding of 32P, not covalent protein phosphorylation. The ability of SCSα to retain its 32P throughout the SDS denaturing gel process was unique over the entire mitochondrial proteome. In vitro studies also revealed a Pi-induced activation of SCS activity by more than 2-fold when mitochondrial extracts and purified BSCS were incubated with mM concentrations of Pi. Since 32P-binding to SCSα was increased in substrate-depleted mitochondria, where matrix Pi concentration is increased, we conclude that SCS activation by Pi-binding represents another mitochondrial target for the Pi-induced activation of oxidative phosphorylation and anaerobic ATP production in energy-limited mitochondria. PMID:19527071

CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

PubMed

Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

2016-08-01

Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs).

PubMed

Recuero-Checa, Maria A; Sharma, Manu; Lau, Constance; Watkins, Paul A; Gaydos, Charlotte A; Dean, Deborah

2016-03-18

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis.

PubMed

Guo, Rey-Ting; Chong, Yeeting E; Guo, Min; Yang, Xiang-Lei

2009-10-16

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.

Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis*

PubMed Central

Guo, Rey-Ting; Chong, Yeeting E.; Guo, Min; Yang, Xiang-Lei

2009-01-01

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed. PMID:19710017

Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate †

PubMed Central

Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D.

2009-01-01

O-succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered Bi Uni Uni Bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct-binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first-half reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the ‘F’ form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogs with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogs tested as none were active except 4-(2-trifluoromethylphenyl)-4-oxobutyric acid which exhibited a 100-fold decrease in kcat/Km. Based on an understanding of OSB-CoA synthetase’s kinetic mechanism and substrate specificity, a reaction intermediate analog of OSB-AMP, 5’-O-(N-(2-trifluoromethylphenyl)-4-oxobutyl) adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of

Aminoacyl-tRNA synthetases database Y2K

PubMed Central

Szymanski, Maciej; Barciszewski, Jan

2000-01-01

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html PMID:10592262

Aminoacyl-tRNA synthetases database Y2K.

PubMed

Szymanski, M; Barciszewski, J

2000-01-01

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloro-plasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

PubMed Central

Hughes, Samantha J; Tanner, Julian A; Hindley, Alison D; Miller, Andrew D; Gould, Ian R

2003-01-01

Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

PubMed Central

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

PubMed

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohda, D.; Kawai, G.; Yokoyama, S.

1987-10-06

The 400-MHz /sup 1/H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS.more » The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche/sup +/ form about the C/sub ..cap alpha../-C/sub ..beta../ bond and the trans form about the C/sub ..beta../-C/sub ..gamma../sub 1// bond. The IleRS-bound L-valine takes the guache/sup -/ form about the C/sub ..cap alpha../-C/sub ..beta../ bond. Thus, the conformation of the IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.« less

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus.

PubMed

Lewis, Amanda L; Cao, Hongzhi; Patel, Silpa K; Diaz, Sandra; Ryan, Wesley; Carlin, Aaron F; Thon, Vireak; Lewis, Warren G; Varki, Ajit; Chen, Xi; Nizet, Victor

2007-09-21

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

PubMed Central

Brattsten, L B; Wilkinson, C F

1975-01-01

1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase. PMID:1004

Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

PubMed

Brattsten, L B; Wilkinson, C F

1975-07-01

1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase.

Dynamics of the active site loops in catalyzing aminoacylation reaction in seryl and histidyl tRNA synthetases.

PubMed

Dutta, Saheb; Kundu, Soumya; Saha, Amrita; Nandi, Nilashis

2018-03-01

Aminoacylation reaction is the first step of protein biosynthesis. The catalytic reorganization at the active site of aminoacyl tRNA synthetases (aaRSs) is driven by the loop motions. There remain lacunae of understanding concerning the catalytic loop dynamics in aaRSs. We analyzed the functional loop dynamics in seryl tRNA synthetase from Methanopyrus kandleri ( mk SerRS) and histidyl tRNA synthetases from Thermus thermophilus ( tt HisRS), respectively, using molecular dynamics. Results confirm that the motif 2 loop and other active site loops are flexible spots within the catalytic domain. Catalytic residues of the loops form a network of interaction with the substrates to form a reactive state. The loops undergo transitions between closed state and open state and the relaxation of the constituent residues occurs in femtosecond to nanosecond time scale. Order parameters are higher for constituent catalytic residues which form a specific network of interaction with the substrates to form a reactive state compared to the Gly residues within the loop. The development of interaction is supported from mutation studies where the catalytic domain with mutated loop exhibits unfavorable binding energy with the substrates. During the open-close motion of the loops, the catalytic residues make relaxation by ultrafast librational motion as well as fast diffusive motion and subsequently relax rather slowly via slower diffusive motion. The Gly residues act as a hinge to facilitate the loop closing and opening by their faster relaxation behavior. The role of bound water is analyzed by comparing implicit solvent-based and explicit solvent-based simulations. Loops fail to form catalytically competent geometry in absence of water. The present result, for the first time reveals the nature of the active site loop dynamics in aaRS and their influence on catalysis.

Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase.

PubMed Central

Meganathan, R; Bentley, R; Taber, H

1981-01-01

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent. PMID:6780515

Structural Biology of Non-Ribosomal Peptide Synthetases

PubMed Central

Miller, Bradley R.; Gulick, Andrew M.

2016-01-01

Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

PubMed Central

Recuero-Checa, Maria A.; Sharma, Manu; Lau, Constance; Watkins, Paul A.; Gaydos, Charlotte A.; Dean, Deborah

2016-01-01

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs. PMID:26988341

A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria.

PubMed

Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel

2015-05-01

Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of Synechocystis GS. A protein-protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction. © 2015 John Wiley & Sons Ltd.

A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors.

PubMed

Esposito, Marta; Szadocka, Sára; Degiacomi, Giulia; Orena, Beatrice S; Mori, Giorgia; Piano, Valentina; Boldrin, Francesca; Zemanová, Júlia; Huszár, Stanislav; Barros, David; Ekins, Sean; Lelièvre, Joel; Manganelli, Riccardo; Mattevi, Andrea; Pasca, Maria Rosalia; Riccardi, Giovanna; Ballell, Lluis; Mikušová, Katarína; Chiarelli, Laurent R

2017-06-09

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with K i values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

Downregulation of acetyl-CoA synthetase 2 is a metabolic hallmark of tumor progression and aggressiveness in colorectal carcinoma.

PubMed

Bae, Jeong Mo; Kim, Jung Ho; Oh, Hyeon Jeong; Park, Hye Eun; Lee, Tae Hun; Cho, Nam-Yun; Kang, Gyeong Hoon

2017-02-01

Acetyl-CoA synthetase-2 is an emerging key enzyme for cancer metabolism, which supplies acetyl-CoA for tumor cells by capturing acetate as a carbon source under stressed conditions. However, implications of acetyl-CoA synthetase-2 in colorectal carcinoma may differ from other malignancies, because normal colonocytes use short-chain fatty acids as an energy source, which are supplied by fermentation of the intestinal flora. Here we analyzed acetyl-CoA synthetase-2 mRNA expression by reverse-transcription quantitative PCR in paired normal mucosa and tumor tissues of 12 colorectal carcinomas, and subsequently evaluated acetyl-CoA synthetase-2 protein expression by immunohistochemistry in 157 premalignant colorectal lesions, including 60 conventional adenomas and 97 serrated polyps, 1,106 surgically resected primary colorectal carcinomas, and 23 metastatic colorectal carcinomas in the liver. In reverse-transcription quantitative PCR analysis, acetyl-CoA synthetase-2 mRNA expression was significantly decreased in tumor tissues compared with corresponding normal mucosa tissues. In acetyl-CoA synthetase-2 immunohistochemistry analysis, all 157 colorectal polyps showed moderate-to-strong expression of acetyl-CoA synthetase-2. However, cytoplasmic acetyl-CoA synthetase-2 expression was downregulated (acetyl-CoA synthetase-2 low expression) in 771 (69.7%) of 1,106 colorectal carcinomas and 21 (91.3%) of 23 metastatic lesions. The colorectal carcinomas with acetyl-CoA synthetase-2-low expression were significantly associated with advanced TNM stage, poor differentiation, and frequent tumor budding. Regarding the molecular aspect, acetyl-CoA synthetase-2-low expression exhibited a tendency of frequent KRT7 expression and decreased KRT20 and CDX2 expression. In survival analysis, acetyl-CoA synthetase-2-low expression was an independent prognostic factor for poor 5-year progression-free survival (hazard ratio, 1.39; 95% confidence interval, 1.08-1.79; P=0.01). In conclusion

The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae.

PubMed

Persuhn, D C; Souza, E M; Steffens, M B; Pedrosa, F O; Yates, M G; Rigo, L U

2000-11-15

The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.

Homology of aspartyl- and lysyl-tRNA synthetases.

PubMed Central

Gampel, A; Tzagoloff, A

1989-01-01

The yeast nuclear gene MSD1 coding for mitochondrial aspartyl-tRNA synthetase has been cloned and sequenced. The identity of the gene is confirmed by the following evidence. (i) The primary structure of the protein derived from the gene sequence is similar to that of the yeast cytoplasmic aspartyl-tRNA synthetase. (ii) In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis. (iii) Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. The primary structures of the cytoplasmic and mitochondrial aspartyl-tRNA synthetases are similar to the yeast cytoplasmic lysyl-tRNA synthetase, suggesting that the two types of synthetases may have a common evolutionary origin. Searches of the current protein banks also have revealed a high degree of sequence similarity of the lysyl-tRNA synthetase to the product of the Escherichia coli herC gene and to the partial sequence of a protein encoded by an unidentified reading frame located adjacent to the E. coli frdA gene. Based on the sequence similarities and the map positions of the herC and frdA loci, we propose herC to be the structural gene of the constitutively expressed lysyl-tRNA synthetase of E. coli and the unidentified reading frame to be the structural gene of the heat-inducible lysyl-tRNA synthetase. Images PMID:2668951

Genetically encoded fluorescent coumarin amino acids

DOEpatents

Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.

2010-10-05

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

Genetically encoded fluorescent coumarin amino acids

DOEpatents

Wang, Jiangyun [San Diego, CA; Xie, Jianming [San Diego, CA; Schultz, Peter G [La Jolla, CA

2012-06-05

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

PubMed Central

Murdeshwar, Maya S.

2012-01-01

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme RelMsm. This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The relMsm knockout strain of M. smegmatis (ΔrelMsm) is expected to show a (p)ppGpp null [(p)ppGpp0] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRelMsm in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response. PMID:22636779

MS_RHII-RSD, a dual-function RNase HII-(p)ppGpp synthetase from Mycobacterium smegmatis.

PubMed

Murdeshwar, Maya S; Chatterji, Dipankar

2012-08-01

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm) knockout strain of M. smegmatis (Δrel(Msm)) is expected to show a (p)ppGpp null [(p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

PubMed

Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

2015-07-15

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity

PubMed Central

2015-01-01

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain. PMID:26120870

Active site nanospace of aminoacyl tRNA synthetase: difference between the class I and class II synthetases.

PubMed

Dutta, Saheb; Choudhury, Kaberi; Banik, Sindrila Dutta; Nandi, Nilashis

2014-03-01

The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the

[ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism].

PubMed

Lemeshko, V V; Kaliman, P A; Belostotskaia, L I; Uchitel', A A

1982-04-01

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.

Unnatural reactive amino acid genetic code additions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander; Cropp, Ashton T; Chin, Jason W; Anderson, Christopher J; Schultz, Peter G

2013-05-21

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander [La Jolla, CA; Cropp, T Ashton [San Diego, CA; Chin, Jason W [Cambridge, GB; Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA

2011-02-15

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.; Anderson, J. Christopher; Schultz, Peter G.

2014-08-26

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Influence of endogenous pyrogen on the cerebral prostaglandin-synthetase system.

PubMed

Ziel, R; Krupp, P

1976-11-15

The biotransformation of arachidonic acid to prostaglandins in vitro is specifically augmented by endogenous pyrogen to a degree depending on the concentration applied, providing that the microsomal fraction of the cerebral cortex is used as prostaglandin-synthetase system. This effect is inhibited by non-steroidal anti-inflammatory agents. These findings are compatible with the hypothesis that prostaglandins might act as mediators of the febrile reaction induced by endogenous pyrogen.

Inhibition of Long Chain Fatty Acyl-CoA Synthetase (ACSL) and Ischemia Reperfusion Injury

PubMed Central

Prior, Allan M.; Zhang, Man; Blakeman, Nina; Datta, Palika; Pham, Hung; Young, Lindon H.; Weis, Margaret T.; Hua, Duy H.

2014-01-01

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion. PMID:24480468

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase*

PubMed Central

Chen, Yaozong; Sun, Yueru; Song, Haigang; Guo, Zhihong

2015-01-01

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes. PMID:26276389

Active role of fatty acid amino acid conjugates in nitrogen metabolism in Spodoptera litura larvae

PubMed Central

Yoshinaga, Naoko; Aboshi, Takako; Abe, Hiroaki; Nishida, Ritsuo; Alborn, Hans T.; Tumlinson, James H.; Mori, Naoki

2008-01-01

Since the first fatty acid amino acid conjugate (FAC) was isolated from regurgitant of Spodoptera exigua larvae in 1997 [volicitin: N-(17-hydroxylinolenoyl)-l-glutamine], their role as elicitors of induced responses in plants has been well documented. However, studies of the biosyntheses and the physiological role of FACs in the insect have been minimal. By using 14C-labeled glutamine, glutamic acid, and linolenic acid in feeding studies of Spodoptera litura larvae, combined with tissue analyses, we found glutamine in the midgut cells to be a major source for biosynthesis of FACs. Furthermore, 20% of the glutamine moiety of FACs was derived from glutamic acid and ammonia through enzymatic reaction of glutamine synthetase (GS). To determine whether FACs improve GS productivity, we studied nitrogen assimilation efficiency of S. litura larvae fed on artificial diets containing 15NH4Cl and glutamic acid. When the diet was enriched with linolenic acid, the nitrogen assimilation efficiency improved from 40% to >60%. In the lumen, the biosynthesized FACs are hydrolyzed to fatty acids and glutamine, which are reabsorbed into tissues and hemolymph. These results strongly suggested that FACs play an active role in nitrogen assimilation in Lepidoptera larva and that glutamine containing FACs in the gut lumen may function as a form of storage of glutamine, a key compound of nitrogen metabolism. PMID:18997016

Glutathione synthetase deficiency: a family report.

PubMed Central

Pejaver, R K; Watson, A H

1994-01-01

Glutathione synthetase deficiency is a rare inborn error of metabolism. Low levels of and at times unstable molecules of glutathione synthetase leads to glutathione deficiency affecting various systems of the body. The inheritance is thought to be of autosomal recessive variety. We diagnosed the condition in a neonate and proceeded to investigate the family. The results are discussed below. PMID:8158601

Pharmacological studies on the TXA2 synthetase inhibitor (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046).

PubMed

Hiraku, S; Taniguchi, K; Wakitani, K; Omawari, N; Kira, H; Miyamoto, T; Okegawa, T; Kawasaki, A; Ujiie, A

1986-07-01

The effects of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046) on thromboxane A2 (TXA2) synthetase in vitro and on experimental animal models of sudden death and cerebral infarction were studied. IC50 values of OKY-046 for the TXA2 synthetase of human, rabbit, dog and guinea pig washed platelets were 0.004, 0.004, 0.26 and 2.4 microM, respectively. OKY-046 at concentrations up to 1 mM, however, did not inhibit prostacyclin (PGI2) synthetase from bovine aorta microsomes or cyclooxygenase and PGE2 isomerase from sheep seminal vesicle microsomes. Similarly, platelet 12-lipoxygenase was not affected by OKY-046. Evidence for a re-direction of arachidonate metabolism from thromboxane synthesis toward PGI2 synthesis was obtained using rat peritoneal cells. Namely, OKY-046 increased PGI2 production accompanied by an inhibition of TXA2 production at a concentration of more than 1 microM. OKY-046 at a dose of 0.1 mg/kg (i.v.) in dogs inhibited the aortic and mesenteric arterial contraction of rabbit induced by the addition of arachidonate to extracorporated blood of the dogs. OKY-046 at a dose of 0.3 mg/kg (i.v.) prevented the arachidonate-induced sudden death and also decreased the incidence of cerebral infarction induced by injection of arachidonate into the internal carotid artery in rabbits. Aspirin also decreased the incidence of cerebral infarction at a dose of 30 mg/kg (i.v.). These results suggest that OKY-046 may be valuable for the treatment of cerebrovascular and cardiovascular diseases associated with vasoconstriction and thrombosis due to TXA2.

Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites☆

PubMed Central

Pham, James S.; Dawson, Karen L.; Jackson, Katherine E.; Lim, Erin E.; Pasaje, Charisse Flerida A.; Turner, Kelsey E.C.; Ralph, Stuart A.

2013-01-01

Aminoacyl-tRNA synthetases are central enzymes in protein translation, providing the charged tRNAs needed for appropriate construction of peptide chains. These enzymes have long been pursued as drug targets in bacteria and fungi, but the past decade has seen considerable research on aminoacyl-tRNA synthetases in eukaryotic parasites. Existing inhibitors of bacterial tRNA synthetases have been adapted for parasite use, novel inhibitors have been developed against parasite enzymes, and tRNA synthetases have been identified as the targets for compounds in use or development as antiparasitic drugs. Crystal structures have now been solved for many parasite tRNA synthetases, and opportunities for selective inhibition are becoming apparent. For different biological reasons, tRNA synthetases appear to be promising drug targets against parasites as diverse as Plasmodium (causative agent of malaria), Brugia (causative agent of lymphatic filariasis), and Trypanosoma (causative agents of Chagas disease and human African trypanosomiasis). Here we review recent developments in drug discovery and target characterisation for parasite aminoacyl-tRNA synthetases. PMID:24596663

Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism

PubMed Central

Madiraju, Anila K.; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W.; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T.; Kibbey, Richard G.; Shulman, Gerald I.

2016-01-01

A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism. PMID:27247419

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

PubMed Central

Knudsen, J

1979-01-01

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue. PMID:574008

Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

PubMed

Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

2014-01-01

Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

PubMed Central

Pérez-Delgado, Carmen M.; García-Calderón, Margarita; Márquez, Antonio J.; Betti, Marco

2015-01-01

It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4 + accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4 + when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4 +. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4 + when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity. PMID:26091523

Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy

NASA Technical Reports Server (NTRS)

D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen

1987-01-01

Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder

PubMed Central

Modi, Hiren R.; Basselin, Mireille; Taha, Ameer Y.; Li, Lei O.; Coleman, Rosalind A.; Bialer, Meir; Rapoport, Stanley I.

2013-01-01

Background Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain acyl-CoA synthetase (Acsl)-4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl-4 catalyzed acylation, and thus have potential anti-BD action. Methods Rat Acsl4-flag protein was expressed in E. coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis-Menten kinetics. Results Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4 mM compared to a published Ki of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect. Conclusions PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients. PMID:23354024

Biosynthetic engineering of nonribosomal peptide synthetases.

PubMed

Kries, Hajo

2016-09-01

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Characteristics of a leucine aminoacyl transfer RNA synthetase from Tritrichomonas augusta.

PubMed

Horner, J; Champney, W S; Samuels, R

1991-04-01

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37 degrees C for 20 min, with protein at 180 micrograms ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

PubMed

Dayan, A; Bertrand, R; Beauchemin, M; Chahla, D; Mamo, A; Filion, M; Skup, D; Massie, B; Jolivet, J

1995-11-20

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Biochemical heterogeneity in glutathione synthetase deficiency.

PubMed Central

Spielberg, S P; Garrick, M D; Corash, L M; Butler, J D; Tietze, F; Rogers, L; Schulman, J D

1978-01-01

Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline. PMID:659603

The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui

Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscentmore » of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.« less

Construction of hybrid peptide synthetases by module and domain fusions

PubMed Central

Mootz, Henning D.; Schwarzer, Dirk; Marahiel, Mohamed A.

2000-01-01

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min-1 and 2.1 min-1. The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides. PMID:10811885

Construction of hybrid peptide synthetases by module and domain fusions.

PubMed

Mootz, H D; Schwarzer, D; Marahiel, M A

2000-05-23

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

PubMed

Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

2016-05-03

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Effects of protopine on blood platelet aggregation. III. Effect of protopine on the metabolic system of arachidonic acid in platelets.

PubMed

Shiomoto, H; Matsuda, H; Kubo, M

1991-02-01

The mode of action of protopine on blood platelet aggregation was investigated in the metabolic system of arachidonic acid and in liberation of platelet activating factor using in vitro experimental models. Protopine inhibited the releases of arachidonic acid and platelet activating factor from platelet membrane phospholipids. Protopine also inhibited the conversion of prostaglandin G2 to thromboxane A2, as well as carboxyheptyl imidazole, a thromboxane synthetase inhibitor. These results indicated that protopine functions both as a phospholipase inhibitor and a thromboxane synthetase inhibitor. It is expected that protopine can be applied for treatment of thrombosis as an antiplatelet drug.

Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs

PubMed Central

Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; Van Voorhis, Wesley C; Hol, Wim G J

2010-01-01

Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active site rearrangement upon histidine binding, but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme’s first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human histidyl-tRNA synthetases. The essentiality of the single histidyl-tRNA synthetase gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference. PMID:20132829

Characterization of cDNAs and genomic DNAs for human threonyl- and cysteinyl-tRNA synthetases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cruzen, M.E.

1993-01-01

Techniques of molecular biology were used to clone, sequence and map two human aminoacyl-tRNA synthetase (aaRS) cDNAs: threonyl-tRNA synthetase (ThrRS) a class II enzyme and cysteinyl-tRNA synthetase (CysRS) a class I enzyme. The predicted protein sequence of human ThrRS is highly homologous to that of lower eukaryotic and prokaryotic ThRSs, particularly in the regions containing the three structural motifs common to all class II synthetases. Signature regions 1 and 2, which characterize the class IIa subgroup (SerRS, ThrRS and HisRS) are highly conserved from bacteria to human. Structural predictions for human ThrRS based on the known structure of the closelymore » related SerRS from E.coli implicate strongly conserved residues in the signature sequences to be important in substrate binding. The amino terminal 100 residues of the deduced amino acid sequence of ThrRS shares structural similarity to SerRS consistent with forming an antiparallel helix implicated in tRNA binding. The 5' untranslated sequence of the human ThrRS gene shares short stretches of common sequence with the gene for hamster HisRS including a binding site for the promoter specific transcription factor sp-1. The deduced amino acid sequence of human CysRS has a high degree of sequence identify to E. coli CysRS. Human CysRS possesses the classic characteristics of a class I synthetase and is most closely related to the MetRS subgroup. The amino terminal half of human CysRS can be modeled as a nucleotide binding fold and shares significant sequence and structural similarity to the other enzymes in this subgroup. The CysRS structural gene (CARS) was mapped to human chromosome 11p15.5 by fluorescent in situ hybridization. CARS is the first aaRS gene to be mapped to chromosome 11. The steady state of both CysRS and ThrRs mRNA were quantitated in several human tissues. Message levels for these enzymes appear to be subjected to differential regulation in different cell types.« less

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

PubMed

Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

2014-03-01

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis. © 2013 Scandinavian Plant Physiology Society.

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

NASA Technical Reports Server (NTRS)

Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

1992-01-01

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera].

PubMed

Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just; Kelve, Merike; Uriz, Maria Jesus

2009-01-01

Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2',5'-oligoadenylate synthetase (OAS). The components of the antiviral 2',5'-oligoadenylate (2-5A) system (OAS, 2'-Phosphodiesterase (2'-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2'-PDE activity, which highlights the probable existence of a premature 2-5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2-5A degrading activity. Upon this finding, two out of three elements forming the 2-5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.

How does Pseudomonas fluorescens avoid suicide from its antibiotic pseudomonic acid?: Evidence for two evolutionarily distinct isoleucyl-tRNA synthetases conferring self-defense.

PubMed

Yanagisawa, Tatsuo; Kawakami, Makoto

2003-07-11

Two isoleucyl-tRNA synthetases (IleRSs) encoded by two distinct genes (ileS1 and ileS2) were identified in pseudomonic acid (mupirocin)-producing Pseudomonas fluorescens. The most striking difference between the two IleRSs (IleRS-R1 and IleRS-R2) is the difference in their abilities to resist pseudomonic acid. Purified IleRS-R2 showed no sensitivity to pseudomonic acid even at a concentration of 5 mm, 105 times higher than the Ki value of IleRS-R1. The amino acid sequence of IleRS-R2 exhibits eukaryotic features that are originally found in eukaryotic proteins. Escherichia coli cells transformed with the ileS2 gene exerted pseudomonic acid resistance more than did those transformed with ileS1. Cells transformed with both genes became almost as resistant as P. fluorescens. These results suggest that the presence of IleRS-R2 could be the major reason why P. fluorescens is intrinsically resistant to the antibiotic. Here we suggest that the evolutionary scenario of the eukaryotic ileS2 gene can be explained by gene acquisition and that the pseudomonic acid producer may have maintained the ileS2 gene to protect itself from pseudomonic acid.

Orthogonal translation components for the in vivo incorporation of unnatural amino acids

DOEpatents

Schultz, Peter G.; Alfonta, Lital; Chittuluru, Johnathan R.; Deiters, Alexander; Groff, Dan; Summerer, Daniel; Tsao, Meng -Lin; Wang, Jiangyun; Wu, Ning; Xie, Jianming; Zeng, Huaqiang; Seyedsayamdost, Mohammad; Turner, James

2015-08-11

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate unnatural amino acid into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Orthogonal translation components for the in vivo incorporation of unnatural amino acids

DOEpatents

Schultz, Peter G.; Xie, Jianming; Zeng, Huaqiang

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

PubMed

Davis, Tony D; Mohandas, Poornima; Chiriac, Maria I; Bythrow, Glennon V; Quadri, Luis E N; Tan, Derek S

2016-11-01

Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate β-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

DOEpatents

Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

2008-07-15

Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli.

PubMed Central

Tobin, M B; Kovacevic, S; Madduri, K; Hoskins, J A; Skatrud, P L; Vining, L C; Stuttard, C; Miller, J R

1991-01-01

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete. Images PMID:1917855

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-03-22

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2008-10-07

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-12-06

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Hemolytic anemia and metabolic acidosis: think about glutathione synthetase deficiency.

PubMed

Ben Ameur, Salma; Aloulou, Hajer; Nasrallah, Fehmi; Kamoun, Thouraya; Kaabachi, Naziha; Hachicha, Mongia

2015-02-01

Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction

USDA-ARS?s Scientific Manuscript database

OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo act...

Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine synthetase C-like protein 2.

PubMed

Lu, Pinyi; Hontecillas, Raquel; Horne, William T; Carbo, Adria; Viladomiu, Monica; Pedragosa, Mireia; Bevan, David R; Lewis, Stephanie N; Bassaganya-Riera, Josep

2012-01-01

Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA), a plant hormone with anti-diabetic and anti-inflammatory effects. The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses. LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates.

Computational Modeling-Based Discovery of Novel Classes of Anti-Inflammatory Drugs That Target Lanthionine Synthetase C-Like Protein 2

PubMed Central

Lu, Pinyi; Hontecillas, Raquel; Horne, William T.; Carbo, Adria; Viladomiu, Monica; Pedragosa, Mireia; Bevan, David R.; Lewis, Stephanie N.; Bassaganya-Riera, Josep

2012-01-01

Background Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA), a plant hormone with anti-diabetic and anti-inflammatory effects. Methodology/Principal Findings The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses. Conclusions/Significance LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates. PMID:22509338

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abramchik, Yu. A.; Timofeev, V. I., E-mail: tostars@mail.ru; Muravieva, T. I.

2017-01-15

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°Ð¡, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample,more » which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.« less

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

NASA Astrophysics Data System (ADS)

Abramchik, Yu. A.; Timofeev, V. I.; Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S.; Kuranova, I. P.

2017-01-01

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus ( T. th HB27) has high thermal stability and shows maximum activity at 75°C, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.

Regulation of a glutamyl-tRNA synthetase by the heme status

PubMed Central

Levicán, Gloria; Katz, Assaf; de Armas, Merly; Núñez, Harold; Orellana, Omar

2007-01-01

Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the C5 pathway. In this route Glu-tRNA is transformed to δ-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase. GluTR is a target of feedback regulation by heme. In Acidithiobacillus ferrooxidans, an acidophilic bacterium that expresses two GluRSs (GluRS1 and GluRS2) with different tRNA specificity, the intracellular heme level varies depending on growth conditions. Under high heme requirement for respiration increased levels of GluRS and GluTR are observed. Strikingly, when intracellular heme is in excess, the cells respond by a dramatic decrease of GluRS activity and the level of GluTR. The recombinant GluRS1 enzyme is inhibited in vitro by hemin, but NADPH restores its activity. These results suggest that GluRS plays a major role in regulating the cellular level of heme. PMID:17360620

Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity

PubMed Central

Lee, Eun-Young; Lee, Hyun-Cheol; Kim, Hyun-Kwan; Jang, Song Yee; Park, Seong-Jun; Kim, Yong-Hoon; Kim, Jong Hwan; Hwang, Jungwon; Kim, Jae-Hoon; Kim, Tae-Hwan; Arif, Abul; Kim, Seon-Young; Choi, Young-Ki; Lee, Cheolju; Lee, Chul-Ho; Jung, Jae U; Fox, Paul L; Kim, Sunghoon; Lee, Jong-Soo; Kim, Myung Hee

2016-01-01

The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/−) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection. PMID:27595231

Structural Switch of Lysyl-tRNA Synthetase Between Translation and Transcription

PubMed Central

Ofir-Birin, Yifat; Fang, Pengfei; Bennett, Steven P.; Zhang, Hui-Min; Wang, Jing; Rachmin, Inbal; Shapiro, Ryan; Song, Jing; Dagan, Arie; Pozo, Jorge; Kim, Sunghoon; Marshall, Alan G.; Schimmel, Paul; Yang, Xiang-Lei; Nechushtan, Hovav; Razin, Ehud; Guo, Min

2013-01-01

SUMMARY Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207-phosphorylation provokes a new conformer of LysRS that inactivates its translational, but activates its transcriptional function. The crystal structure of an MSC sub-complex established that LysRS is held in the MSC by binding to the N-terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap4A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription. PMID:23159739

Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus.

PubMed

Oka, Manami; Takegawa, Kaoru; Kimura, Yoshio

2015-08-01

Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn(2+) for the synthesis of Ap4A. Surprisingly, Zn(2+) inhibited enzyme activity in the presence of Mn(2+). When incubated with ATP, Mn(2+), lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported. Copyright © 2015 Elsevier Inc. All rights reserved.

Enhancement of lysyl-tRNA synthetase activity in the Enterobacteriaceae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hickey, E.W.; Hirshfield, I.

1987-05-01

Lysyl-tRNA synthetase (LRS) in E. coli is coded by two genes, one constitutive, and the other inducible; the latter is a cell stress protein. To determine if this system is wide spread in prokaryotes, the inducibility of LRS was first tested in eight members of the Enterobacteriaceae using cultural conditions known to induce the enzyme in E. coli K-12. Uninduced control cultures were grown to an O.D. of 0.2 at 580 nm in a supplemented minimal medium (SMM), pH 7.0 at 37/sup 0/C. Induction stimuli include: growth in SMM with 3mM Gly-L-Leu; growth in SMM as above, but with themore » initial pH adjusted to 5.0; or growth in Difco AC Broth to early stationary phase with a concomitant drop in the pH of the medium below 5.5. LRS activity was assayed in whole-cell sonic extracts by the aminoacylation of crude E. coli tRNA by /sup 14/C-lysine at pH 7.8 for three minutes. When E. aerogenes, K. pneumoniae, C. freundii, and S. typhimurium were grown in AC Broth, LRS activity was enhanced 2 to 4 fold. The enzyme is induced 2 to 4 fold in C. freundii and S. typhimurium upon growth at pH 5.0, whereas E. coli, K.; pneumoniae, and E. aerogenes show only a 1.5 fold induction. The peptide Gly-L-Leu enhanced LRS activity only in E. coli. LRS was not found to be inducible in S. marcescens, M. morganii, P. mirabilis, or P. vulgaris by any of the stimuli.« less

Inhibition of Carbamyl Phosphate Synthetase-I and Glutamine Synthetase by Hepatotoxic Doses of Acetaminophen in Mice

PubMed Central

Gupta, Sanjiv; Rogers, Lynette K.; Taylor, Sarah K.; Smith, Charles V.

2016-01-01

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS–PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question. PMID:9344900

Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru; Abramchik, Yu. A.; Zhukhlistova, N. E.

2016-01-15

Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sitesmore » were shown to be identical in both proteins.« less

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

PubMed Central

Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

2016-01-01

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum.

PubMed

Chen, Zhuo; Luo, Ling; Chen, Runfa; Hu, Hanhua; Pan, Yufang; Jiang, Haibo; Wan, Xia; Jin, Hu; Gong, Yangmin

2018-03-01

N ε -lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD + -dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Glutamine Synthetase Isoenzymes in the Green Soil Alga Stichococcus bacillaris Naeg.

PubMed

Ahmad, I; Hellebust, J A

1987-02-01

Two forms of glutamine synthetase (GS(1) and GS(2)) have been separated from cells of Stichococcus bacillaris by fast protein liquid chromatography. The activities of the two isoenzymes were influenced by the composition of the media employed; thiol reagents were essential for stabilizing GS(2) but they suppressed GS(1) activity. The activity of each isoenzyme was, therefore, determined following separate purification procedures. Growth conditions influenced both isoenzymes; GS(2) showed maximum activity under photoautotrophic conditions, whereas GS(1) showed maximum activity under heterotrophic conditions.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds.

PubMed

Gaufichon, Laure; Marmagne, Anne; Belcram, Katia; Yoneyama, Tadakatsu; Sakakibara, Yukiko; Hase, Toshiharu; Grandjean, Olivier; Clément, Gilles; Citerne, Sylvie; Boutet-Mercey, Stéphanie; Masclaux-Daubresse, Céline; Chardon, Fabien; Soulay, Fabienne; Xu, Xiaole; Trassaert, Marion; Shakiebaei, Maryam; Najihi, Amina; Suzuki, Akira

2017-08-01

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter Ca MV 35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

PubMed Central

Novoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marín-García, Patricia; Azcárate, Isabel G.; Bautista, José M.; Mirando, Adam C.; Francklyn, Christopher S.; Varon, Sònia; Royo, Miriam; Cortés, Alfred; Ribas de Pouplana, Lluís

2014-01-01

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo. PMID:25489076

Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin.

PubMed

Khan, Sameena; Sharma, Arvind; Belrhali, Hassan; Yogavel, Manickam; Sharma, Amit

2014-06-01

Malaria parasites inevitably develop drug resistance to anti-malarials over time. Hence the immediacy for discovering new chemical scaffolds to include in combination malaria drug therapy. The desirable attributes of new chemotherapeutic agents currently include activity against both liver and blood stage malaria parasites. One such recently discovered compound called cladosporin abrogates parasite growth via inhibition of Plasmodium falciparum lysyl-tRNA synthetase (PfKRS), an enzyme central to protein translation. Here, we present crystal structure of ternary PfKRS-lysine-cladosporin (PfKRS-K-C) complex that reveals cladosporin's remarkable ability to mimic the natural substrate adenosine and thereby colonize PfKRS active site. The isocoumarin fragment of cladosporin sandwiches between critical adenine-recognizing residues while its pyran ring fits snugly in the ribose-recognizing cavity. PfKRS-K-C structure highlights ample space within PfKRS active site for further chemical derivatization of cladosporin. Such derivatives may be useful against additional human pathogens that retain high conservation in cladosporin chelating residues within their lysyl-tRNA synthetase.

Electron Transport Controls Glutamine Synthetase Activity in the Facultative Heterotrophic Cyanobacterium Synechocystis sp. PCC 6803.

PubMed Central

Reyes, J. C.; Crespo, J. L.; Garcia-Dominguez, M.; Florencio, F. J.

1995-01-01

Glutamine synthetase (GS) from Synechocystis sp. PCC 6803 was inactivated in vivo by transferring cells from light to darkness or by incubation with the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but not with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. Addition of glucose prevented both dark and 3-(3,4-dichlorophenyl)-1,1-dimethylurea GS inactivation. In a Synechocystis psbE-psbF mutant (T1297) lacking photosystem II, glucose was required to maintain active GS, even in the light. However, in nitrogen-starved T1297 cells the removal of glucose did not affect GS activity. The fact that dark-inactivated GS was reactivated in vitro by the same treatments that reactivate the ammonium-inactivated GS points out that both nitrogen metabolism and redox state of the cells lead to the same molecular regulatory mechanism in the control of GS activity. Using GS antibodies we detected that dark-inactivated GS displayed a different electrophoretic migration with respect to the active form in nondenaturing polyacrylamide gel electrophoresis but not in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possible pathway to modulate GS activity by the electron transport flow in Synechocystis cells is discussed. PMID:12228640

Drosophila selenophosphate synthetase 1 regulates vitamin B6 metabolism: prediction and confirmation

PubMed Central

2011-01-01

Background There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis in Drosophila. The function of SPS1, however, has not been elucidated. Results Differentially expressed genes in Drosophila SL2 cells were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage at which megamitochondria were not formed (day 3) after SPS1 knockdown. Interestingly, genes related to defense and amino acid metabolism were affected at a later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by SPS1 knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by SPS1 knockdown and the formation of megamitochondria, the major phenotypic change observed by SPS1 knockdown. Conclusions These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities. PMID:21864351

Liver glutamine synthetase activity is markedly reduced in chronic ethanol-fed micropigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olin, K.L.; Zidenberg-Cherr, S.; Villanueva, J.

1992-02-26

The authors have reported that chronic ethanol (Et) feeding in the micropig results in changes in antioxidant defense including reductions in liver CuZnSOD and GSHPX activities, in vitamin E and A levels, and increases in liver MnSOD activity. Despite these alterations, liver mitochondria (mit) and microsome (mic) TBARS were lower in Et-fed than control (C) pigs. The significance of lower TBARS is unclear since the saturated to PUFA ratio was higher in mit and mic from Et than from C pigs. Thus in the current study they measured a non-lipid target of oxidative damage. Glutamine synthetase (GS) activity was measuredmore » as this protein is an excellent marker for oxidative damage due to the sensitivity of histidine residues to free radicals at the active site. Micropigs were fed high PUFA diets containing 40% of kcals as either Et or cornstarch (C) and 34% of kcals as corn oil. After 12 mo pigs were killed and livers removed. Fatty infiltration, inflammation and necrosis were observed in livers from Et pigs by 5 mo; collagen infiltration was apparent in 2 pigs by 12 mo. Et pigs had GS activities that were 90% lower than C pigs. The finding that liver Mn levels were higher in Et than in C pigs suggests that the low GS activity is not due to a reduction in Mn availability, although a shift in the distribution of Mn from GS to MnSOD may be involved. These data support the idea that chronic Et feeding is associated with oxidative damage and underscore the need to evaluate non-lipid targets as markers for oxidative damage.« less

Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

PubMed Central

Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

2009-01-01

AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG

PubMed Central

Mori, Giorgia; Chiarelli, Laurent R.; Esposito, Marta; Makarov, Vadim; Bellinzoni, Marco; Hartkoorn, Ruben C.; Degiacomi, Giulia; Boldrin, Francesca; Ekins, Sean; de Jesus Lopes Ribeiro, Ana Luisa; Marino, Leonardo B.; Centárová, Ivana; Svetlíková, Zuzana; Blaško, Jaroslav; Kazakova, Elena; Lepioshkin, Alexander; Barilone, Nathalie; Zanoni, Giuseppe; Porta, Alessio; Fondi, Marco; Fani, Renato; Baulard, Alain R.; Mikušová, Katarína; Alzari, Pedro M.; Manganelli, Riccardo; de Carvalho, Luiz Pedro S.; Riccardi, Giovanna; Cole, Stewart T.; Pasca, Maria Rosalia

2015-01-01

Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target. PMID:26097035

Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evdokimov, Artem G.; Mekel, Marlene; Hutchings, Kim

2008-07-08

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality -- they only diffracted X-rays to 3--5 {angstrom} resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 {angstrom} or better. This methodology is discussed andmore » contrasted with the more traditional domain truncation approach.« less

Beauvericin synthetase contains a calmodulin binding motif in the entomopathogenic fungus Beauveria bassiana.

PubMed

Kim, Jiyoung; Sung, Gi-Ho

2018-03-19

Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca 2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca 2+ signals are partially decoded by Ca 2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca 2+ -dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca 2+ . In addition, in vitro CaM-binding assay revealed that the His-tagged BbBEAS (amino acids 2421-2538) binds to CaM in a Ca 2+ -dependent manner. Therefore, this work suggests that BbBEAS is a novel CaM-binding protein in B. bassiana.

The function of lysyl-tRNA synthetase and Ap4A as signaling regulators of MITF activity in FcepsilonRI-activated mast cells.

PubMed

Lee, Yu-Nee; Nechushtan, Hovav; Figov, Navah; Razin, Ehud

2004-02-01

The involvement of microphthalmia transcription factor (MITF) in the function of mast cells, melanocytes, and osteoclasts has recently started to be investigated in depth. In a previous study, we found Hint to be associated with MITF in mast cells and showed that it suppresses MITF's transcriptional activity. Here, we have found that lysyl-tRNA synthetase (LysRS) is also associated with MITF and forms a multicomplex with MITF and Hint. We have also shown that Ap4A, an endogenous molecule consisting of two adenosine linked by four phosphate which is known to be synthesized by LysRS, is accumulated intracellularily above 700 microM in IgE-Ag-activated mast cells, binds to Hint, liberates MITF, and thus leads to the activation of MITF-dependent gene expression. This implies that LysRS plays a key role via Ap4A as an important signaling molecule in MITF transcriptional activity.

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

PubMed

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-06-18

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

PubMed

Kondo, Ayano; Yamamoto, Shogo; Nakaki, Ryo; Shimamura, Teppei; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Yoshida, Tetsuo; Aburatani, Hiroyuki; Osawa, Tsuyoshi

2017-02-28

Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

PubMed

Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

2014-01-01

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

Aminoacyl-tRNA synthetase deficiencies in search of common themes.

PubMed

Fuchs, Sabine A; Schene, Imre F; Kok, Gautam; Jansen, Jurriaan M; Nikkels, Peter G J; van Gassen, Koen L I; Terheggen-Lagro, Suzanne W J; van der Crabben, Saskia N; Hoeks, Sanne E; Niers, Laetitia E M; Wolf, Nicole I; de Vries, Maaike C; Koolen, David A; Houwen, Roderick H J; Mulder, Margot F; van Hasselt, Peter M

2018-06-06

Pathogenic variations in genes encoding aminoacyl-tRNA synthetases (ARSs) are increasingly associated with human disease. Clinical features of autosomal recessive ARS deficiencies appear very diverse and without apparent logic. We searched for common clinical patterns to improve disease recognition, insight into pathophysiology, and clinical care. Symptoms were analyzed in all patients with recessive ARS deficiencies reported in literature, supplemented with unreported patients evaluated in our hospital. In literature, we identified 107 patients with AARS, DARS, GARS, HARS, IARS, KARS, LARS, MARS, RARS, SARS, VARS, YARS, and QARS deficiencies. Common symptoms (defined as present in ≥4/13 ARS deficiencies) included abnormalities of the central nervous system and/or senses (13/13), failure to thrive, gastrointestinal symptoms, dysmaturity, liver disease, and facial dysmorphisms. Deep phenotyping of 5 additional patients with unreported compound heterozygous pathogenic variations in IARS, LARS, KARS, and QARS extended the common phenotype with lung disease, hypoalbuminemia, anemia, and renal tubulopathy. We propose a common clinical phenotype for recessive ARS deficiencies, resulting from insufficient aminoacylation activity to meet translational demand in specific organs or periods of life. Assuming residual ARS activity, adequate protein/amino acid supply seems essential instead of the traditional replacement of protein by glucose in patients with metabolic diseases.

Genetics Home Reference: holocarboxylase synthetase deficiency

MedlinePlus

... holocarboxylase synthetase deficiency Orphanet: Multiple carboxylase deficiency Screening, Technology, and Research in Genetics Virginia Department of Health (PDF) Patient Support and Advocacy Resources (3 links) Children Living with Inherited Metabolic Diseases Organic Acidemia Association ...

Nucleotide synthetase ribozymes may have emerged first in the RNA world

PubMed Central

Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

2007-01-01

Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells. PMID:17878321

Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

PubMed Central

Hoff-Risseti, Caroline; Dörr, Felipe Augusto; Schaker, Patricia Dayane Carvalho; Pinto, Ernani; Werner, Vera Regina; Fiore, Marli Fatima

2013-01-01

The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins. PMID:24015317

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings 1

PubMed Central

Lee, Sung-Chul; West, Charles A.

1981-01-01

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis. PMID:16661728

Treatment of renal colic by prostaglandin synthetase inhibitors and avafortan (analgesic antispasmodic).

PubMed

el-Sherif, A E; Foda, R; Norlen, L J; Yahia, H

1990-12-01

In a study of the pain-relieving effect of 3 drugs commonly used to treat acute renal colic in this hospital, intravenous indomethacin and intramuscular diclofenac (prostaglandin synthetase inhibitors) were compared with intravenous Avafortan (analgesic antispasmodic). As first-line analgesics, prostaglandin synthetase inhibitors, if given intravenously, offer an effective alternative to Avafortan. Of 145 patients studied, 32 required a second injection for complete relief of pain. Administering a second dose of prostaglandin synthetase inhibitors resulted in equally significant pain relief rate even though the route was intramuscular.

Interferon action: two (2'-5')(A)n synthetases specified by distinct mRNAs in Ehrlich ascites tumor cells treated with interferon.

PubMed

St Laurent, G; Yoshie, O; Floyd-Smith, G; Samanta, H; Sehgal, P B; Lengyel, P

1983-05-01

(2'-5')(A)n synthetase and RNAase L (a latent endoribonuclease) are among the mediators of interferon action. The product of (2'-5')(A)n synthetase (i.e., (2'-5')(A)n) binds, and thereby activates RNAase L. Interferons induce in Ehrlich ascites tumor (EAT) cells two mRNAs (sizes 1.5 kb and 3.8 kb), which can be translated in Xenopus oocytes into (2'-5')(A)n synthetases of 20,000 to 30,000 daltons and 85,000 to 100,000 daltons, respectively. (2'-5')(A)n synthetases of corresponding sizes are induced by interferons in EAT cells. In the cell extract the bulk of the larger enzyme is in the cytoplasmic fraction, and the bulk of the smaller one in the nuclear fraction. The only known function of (2'-5')(A)n is the activation of RNAase L, and RNAase L can be selectively crosslinked to a (2'-5')(A)n derivative in a cytoplasmic extract from EAT cells. The same (2'-5')(A)n derivative can be crosslinked to several proteins in the nuclear extract of EAT cells, and some of these proteins are induced by interferon.

Glutamine Synthetase Isoenzymes in the Green Soil Alga Stichococcus bacillaris Naeg. 1

PubMed Central

Ahmad, Iftikhar; Hellebust, Johan A.

1987-01-01

Two forms of glutamine synthetase (GS1 and GS2) have been separated from cells of Stichococcus bacillaris by fast protein liquid chromatography. The activities of the two isoenzymes were influenced by the composition of the media employed; thiol reagents were essential for stabilizing GS2 but they suppressed GS1 activity. The activity of each isoenzyme was, therefore, determined following separate purification procedures. Growth conditions influenced both isoenzymes; GS2 showed maximum activity under photoautotrophic conditions, whereas GS1 showed maximum activity under heterotrophic conditions. PMID:16665232

Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene.

PubMed

Chen, Shun-Jia; Wu, Yi-Hua; Huang, Hsiao-Yun; Wang, Chien-Chia

2012-01-01

Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.

Localization of Carbamoylphosphate Synthetase and Aspartate Carbamoyltransferase in Chloroplasts

PubMed Central

Shibata, Hitoshi; Ochiai, Hideo; Sawa, Yoshihiro; Miyoshi, Shoji

1986-01-01

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed. PMID:16664566

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

PubMed Central

Theron, A.; Roth, R. L.; Hoppe, H.; Parkinson, C.; van der Westhuyzen, C. W.; Stoychev, S.; Wiid, I.; Pietersen, R. D.; Baker, B.

2017-01-01

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay. PMID:28972974

Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii.

PubMed

van Rooyen, Jason M; Hakimi, Mohamed-Ali; Belrhali, Hassan

2015-06-01

Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex. Copyright © 2015 Elsevier Inc. All rights reserved.

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.

PubMed Central

Kwon, O; Bhattacharyya, D K; Meganathan, R

1996-01-01

The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular mass of 49 kDa. Thus, the enzyme is a homotetramer. The enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 30 to 40 degrees C. The Km values for OSB, ATP, and CoA were 16, 73.5, and 360 microM, respectively. Of the various metal ions tested, Mg2+ was found to be the most effective in stimulating the enzyme activity. Studies with substrate analogs showed that neither benzoic acid nor benzoylpropionic acid (succinylbenzene) is a substrate for the enzyme. Thus, it appears that both the benzoyl carboxyl group and the succinyl side chain are required for activation of the aliphatic carboxyl group. PMID:8955296

[Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency].

PubMed

Pogorelova, T N; Gunko, V O; Linde, V A

2014-01-01

Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase

PubMed Central

Tang, Yi; Wang, Pin; Van Deventer, James A.; Link, A. James; Tirrell, David A.

2011-01-01

A leucine analog containing a ketone has been incorporated into proteins in E. coli. Only E. coli strains overexpressing an editing-deficient leucyl-tRNA synthetase were capable of synthesizing proteins with the aliphatic ketone amino acid. Modification of ketone-containing proteins under mild conditions has been demonstrated. PMID:19670197

Anthranilate-Activating Modules from Fungal Nonribosomal Peptide Assembly Lines†

PubMed Central

Ames, Brian D.; Walsh, Christopher T.

2010-01-01

Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted β-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N. fischeri. This establishes an anthranilate adenylation domain code for fungal NRPS and should facilitate detection and cloning of gene clusters for benzodiazepine- and quinazoline-containing polycyclic alkaloids with a wide range of biological activities. PMID:20225828

Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

PubMed

Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

2015-11-01

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

The chicken 2'-5' oligoadenylate synthetase A inhibits the replication of West Nile virus.

PubMed

Tag-El-Din-Hassan, Hassan T; Sasaki, Nobuya; Moritoh, Kanako; Torigoe, Daisuke; Maeda, Akihiko; Agui, Takashi

2012-08-01

West Nile virus (WNV) is a pathogen to cause West Nile encephalitis when the infection occurs in the brain. Previous studies in mice identified the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene as a determining factor for resistance to WNV infection. In addition, it has been suggested that human OAS1 and OASL are associated with the resistance to the WNV infection. WNV is maintained in nature through a complex life cycle involving wildbirds and mosquitoes. Birds are not only susceptible to the WNV, but also act as reservoir hosts, thus participating in the spread of the disease. It has previously been reported that chicken OASL possesses the oligoadenylate synthetase activity. However, until now the antiviral activity of chicken OASL has not been determined. In this study, we investigated the putative antiviral activity of chicken OASL by ectopic expression of this enzyme in mammalian cells and then infecting these cells with WNV replicon. We demonstrate that chicken OASL has an antiviral activity against the WNV. This is the first report to show that chicken OASL is associated with the resistance to the WNV infection.

Characterization of the Polymyxin D Synthetase Biosynthetic Cluster and Product Profile of Paenibacillus polymyxa ATCC 10401.

PubMed

Galea, Charles A; Han, Meiling; Zhu, Yan; Roberts, Kade; Wang, Jiping; Thompson, Philip E; L, Jian; Velkov, Tony

2017-05-26

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-α,γ-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D 1 and D 2 but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D 1 and D 2 exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser 3 polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.

Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases.

PubMed

Borgford, T J; Gray, T E; Brand, N J; Fersht, A R

1987-11-17

Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

PubMed Central

Zhang, Yan; Wang, Lei; Schultz, Peter G.; Wilson, Ian A.

2005-01-01

The Methanococcus jannaschii tRNATyr/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-l-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 Å, respectively, for comparison with the published structure of TyrRS complexed with tRNATyr and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257–263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through π-stacking and hydrogen bonding interactions. Loop 133–143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNATyr. Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133–143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over l-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids. PMID:15840835

Holocarboxylase synthetase deficiency: novel clinical and molecular findings.

PubMed

Tammachote, R; Janklat, S; Tongkobpetch, S; Suphapeetiporn, K; Shotelersuk, V

2010-07-01

Multiple carboxylase deficiency (MCD) is an autosomal recessive metabolic disorder caused by defective activity of biotinidase or holocarboxylase synthetase (HLCS) in the biotin cycle. Clinical symptoms include skin lesions and severe metabolic acidosis. Here, we reported four unrelated Thai patients with MCD, diagnosed by urine organic acid analysis. Unlike Caucasians, which biotinidase deficiency has been found to be more common, all of our four Thai patients were affected by HLCS deficiency. Instead of the generally recommended high dose of biotin, our patients were given biotin at 1.2 mg/day. This low-dose biotin significantly improved their clinical symptoms and stabilized the metabolic state on long-term follow-up. Mutation analysis by polymerase chain reaction-sequencing of the entire coding region of the HLCS gene revealed the c.1522C>T (p.R508W) mutation in six of the eight mutant alleles. This suggests it as the most common mutation in the Thai population, which paves the way for a rapid and unsophisticated diagnostic method for the ethnic Thai. Haplotype analysis revealed that the c.1522C>T was on three different haplotypes suggesting that it was recurrent, not caused by a founder effect. In addition, a novel mutation, c.1513G>C (p.G505R), was identified, expanding the mutational spectrum of this gene.

An Iterative, Bimodular Nonribosomal Peptide Synthetase that Converts Anthranilate and Tryptophan into Tetracyclic Asperlicins

PubMed Central

Gao, Xue; Jiang, Wei; Jiménez-Osés, Gonzalo; Choi, Moon Seok; Houk, Kendall N.; Tang, Yi; Walsh, Christopher T.

2013-01-01

The bimodular 276 kDa nonribosomal peptide synthetase AspA from Aspergillus alliaceus, heterologously expressed in Saccharomyces cerevisiae, converts tryptophan and two molecules of the aromatic β-amino acid anthranilate (Ant) into a pair of tetracyclic peptidyl alkaloids asperlicin C and D in a ratio of 10:1. The first module of AspA activates and processes two molecules of Ant iteratively to generate a tethered Ant-Ant-Trp-S-enzyme intermediate on module two. Release is postulated to involve tandem cyclizations, in which the first step is the macrocyclization of the linear tripeptidyl-S-enzyme, by the terminal condensation (CT) domain to generate the regioisomeric tetracyclic asperlicin scaffolds. Computational analysis of the transannular cyclization of the 11-membered macrocyclic intermediate shows that asperlicin C is the kinetically favored product due to the high stability of a conformation resembling the transition state for cyclization, while asperlicin D is thermodynamically more stable. PMID:23890005

Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

PubMed Central

Donnelly, M. Lauren; Fimlaid, Kelly A.

2016-01-01

ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

PubMed

Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

2015-12-18

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus

DOE PAGES

Scott, Joseph W.; Poole, Farris L.; Adams, Michael W. W.

2014-01-01

Tmore » he hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of AP. ACS1 and ACS2 were previously purified from P. furiosus and have α 2 β 2 structures but the genome contains genes encoding three additional α -subunits. he ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α 2 β 2 isoenzymes. he α -subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. he β -subunit determined preference for adenine or guanine nucleotides. he GP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GP for GP-dependent phosphoenolpyruvate carboxykinase and for other GP-dependent processes. ranscriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both AP and GP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the hermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.« less

Tissue-specific changes of glutamine synthetase activity in oats after rhizosphere infestation by Pseudomonas syringae pv. tabaci. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knight, T.J.; Temple, S.; Sengupta-Gopalan, C.

1996-05-15

Oats (Avena sativa L. lodi) tolerant of rhizosphere infestation by Pseudomonas syringae pv. tabaci when challenged by the pathogen experience tissue-specific alterations of ammonia assimilatory capabilities. Altered ammonia assimilatory potentials between root and leaf tissue result from selective inactivation of glutamine synthetase (GS) by the toxin Tabtoxinine-B-lactam (TBL). Root GS is sensitive and leaf GSs are resistant to TBL inactivation. With prolonged challenge by the pathogen root GS activity decreases but leaf GS specific activity increase. Higher leaf GS activity is due to decreased rates of degradation rather than increased GS synthesis. Higher leaf GS activity and elevated levels ofmore » GS polypeptide appear to result from a limited interaction between GS and TBL leading to the accumulation of a less active but more stable GS holoenzyme. Tolerant challenged oats besides surviving rhizosphere infestation, experience enhanced growth. A strong correlation exists between leaf GS activity and whole plant fresh weight, suggesting that tissue-specific changes in ammonia assimilatory capability provides the plant a more efficient mechanism for uptake and utilization of nitrogen.« less

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.

PubMed

Mitchell, Carter A; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2012-04-17

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.

Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis†

PubMed Central

Lu, Xuequan; Zhang, Huaning; Tonge, Peter J.; Tan, Derek S.

2008-01-01

Menaquinone (vitamin K2) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC50 value of 5.7 μM. PMID:18762421

Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus

PubMed Central

Shiyan, Anna; Thompson, Melanie; Köcher, Saskia; Tausendschön, Michaela; Santos, Helena; Hänelt, Inga; Müller, Volker

2014-01-01

Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated. PMID:24782854

Fatty acid-producing hosts

DOEpatents

Pfleger, Brian F; Lennen, Rebecca M

2013-12-31

Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

Aspirin, protein transacetylation and inhibition of prostaglandin synthetase in the kidney

PubMed Central

Caterson, Robyn J.; Duggin, Geoffrey G.; Horvath, John; Mohandas, Janardanan; Tiller, David

1978-01-01

1 The effect of aspirin on the kidney has been investigated in mice and rabbits. [Acetyl-14C]-aspirin was administered intraperitoneally in doses ranging from subtherapeutic to toxic. The degree of acetylation of protein was determined by the radioactivity remaining on protein precipitates of renal cortex and medulla after sequential washing designed to remove non-covalently bound material. Controls were established, by the use of [carboxyl-14C]-aspirin. 2 The acetyl-14C residue was bound to renal proteins in a linear manner in increasing amounts with increasing dosage up to 100 mg/kg. The [carboxyl-14C]-aspirin was not bound and thus the salicylate portion of the molecule was not bound covalently to the renal protein. The time course of the acetylation was rapid, consistent with the rate of aspirin absorption. The disappearance of acetylated protein was slow, with a T1/2 of 112.5 h in the renal cortex, and 129.5 h in the renal medulla. 3 Differential centrifugation, Sephadex chromatography and gel electrophoresis were carried out on tissue homogenates to determine the site of acetylation. The acetylation was greatest in the microsomal fraction, although all protein fractions showed some degree of acetylation. 4 The prostaglandin synthetase activity of a particulate preparation from rabbit kidney was determined by a spectrophotometric assay of malondialdehyde formation. Aspirin (10 mg/kg, i.v.) significantly inhibited prostaglandin synthetase in the renal cortex and medulla. 5 Aspirin and renal proteins undergo a transacetylation reaction resulting in stable acetylated protein, with acetylation being greatest in the microsomal fraction. Aspirin has been shown to inhibit prostaglandin synthetase and this could lead to functional impairment of the tissue. PMID:102389

Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

MedlinePlus

... synthetase I. This enzyme participates in the urea cycle, which is a sequence of biochemical reactions that occurs in liver cells. The urea cycle processes excess nitrogen, generated when protein is broken ...

Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

PubMed

Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

2016-06-27

This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.

PubMed

Boczonadi, Veronika; Meyer, Kathrin; Gonczarowska-Jorge, Humberto; Griffin, Helen; Roos, Andreas; Bartsakoulia, Marina; Bansagi, Boglarka; Ricci, Giulia; Palinkas, Fanni; Zahedi, René P; Bruni, Francesco; Kaspar, Brian; Lochmüller, Hanns; Boycott, Kym M; Müller, Juliane S; Horvath, Rita

2018-06-15

The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition.

The production of Multiple Small Peptaibol Families by Single 14-Module Peptide Synthetases in Trichoderma/Hypocrea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Degenkolb, Thomas; Aghchehb, Razieh Karimi; Dieckmann, Ralf

2012-03-01

The most common peptaibibiotic structures are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of 3 Trichoderma strains of the major clades reveal the presence of up to 3 types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid adding modules. We here provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina) and T. atroviride produces both 11- and 14- residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis ofmore » their amino acid activating domains and modules. The structures of these peptides may be predicted from the gene structures and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 new sequences), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.« less

tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry

PubMed Central

Carter, Charles W.; Wolfenden, Richard

2016-01-01

abstract The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology. PMID:26595350

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations.

PubMed

Thompson, Damien; Lazennec, Christine; Plateau, Pierre; Simonson, Thomas

2008-05-15

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged

Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

PubMed Central

Olson, N J; Marchin, G L

1984-01-01

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly. PMID:6374167

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

PubMed Central

Bhattacharyya, D K; Kwon, O; Meganathan, R

1997-01-01

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme. PMID:9324253

Role of peroxynitrite and poly (ADP-ribosyl) synthetase activation in cardiovascular derangement induced by zymosan in the rat.

PubMed

Cuzzocrea, S; Zingarelli, B; Caputi, A P

1998-01-01

Peritoneal administration of zymosan in the rat induced a severe inflammatory process characterised by an increase in the plasma levels of nitrite and nitrate, stable metabolites of nitric oxide (NO) and in the levels of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, at 18 hours zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the aorta of zymosan-shocked rats. In ex vivo experiments, thoracic aorta rings of zymosan-treated rats showed a reduced contraction to noradrenaline and reduced responsiveness to the relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). Treatment of zymosan-shocked rats with 3-aminobenzamide or Nicotinamide, inhibitors of poly ADP-ribosil synthetase (PARS) activity reduced the production of peroxynitrite and significantly prevented the cardiovascular dysfunction. Our data suggest that peroxynitrite and PARS activation play a role in the zymosan-induced cardiovascular derangements in the rat.

Overexpression of PGC-1α increases peroxisomal activity and mitochondrial fatty acid oxidation in human primary myotubes.

PubMed

Huang, Tai-Yu; Zheng, Donghai; Houmard, Joseph A; Brault, Jeffrey J; Hickner, Robert C; Cortright, Ronald N

2017-04-01

Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m 2 ; n = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes ( PEXs ) responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1- 14 C]palmitic acid and [1- 14 C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, 1 ) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated ( P < 0.05), 2 ) PGC-1α , PMP70 , key PEXs , and peroxisomal β-oxidation mRNA expression levels were significantly upregulated ( P < 0.05), and 3 ) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed ( P < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells. Copyright © 2017 the American Physiological Society.

Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export

PubMed Central

Azad, Abul K.; Stanford, David R.; Sarkar, Srimonti; Hopper, Anita K.

2001-01-01

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 2000a) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export. PMID:11359929

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export.

PubMed

Azad, A K; Stanford, D R; Sarkar, S; Hopper, A K

2001-05-01

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander [La Jolla, CA; Cropp, T Ashton [Bethesda, MD; Chin, Jason W [Cambridge, GB; Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA

2011-08-09

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNAsyn-thetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design.

PubMed

Krajewski, Wojciech W; Collins, Ruairi; Holmberg-Schiavone, Lovisa; Jones, T Alwyn; Karlberg, Tobias; Mowbray, Sherry L

2008-01-04

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

PubMed

Abbott, Jamie A; Guth, Ethan; Kim, Cindy; Regan, Cathy; Siu, Victoria M; Rupar, C Anthony; Demeler, Borries; Francklyn, Christopher S; Robey-Bond, Susan M

2017-07-18

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA His binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

The role of adrenal hormones in the response of glutamine synthetase to fasting in adult and old rats.

PubMed

Mezzarobba, V; Torrent, A; Leydier, I; Alles, S; Brajon, B; Mignon, M; Attaix, D; Meynial-Denis, D

2003-12-01

During fasting, skeletal muscle exports increased amounts of glutamine (Gln) while increasing the production of this amino acid by glutamine synthetase (GS) in order to maintain the intramuscular Gln pool. Glucocorticoid hormones are believed to be the principal mediators of GS induction during stress conditions. The aim of this study was to evaluate (1) the effect of fasting on GS activity and expression in skeletal muscle during aging and consequently, (2) the role of glucocorticoids in fasting-induced GS activity. Male Wistar rats (6-, 22-month old) were fasted for 5 days and both the activity and expression of GS were measured in tibialis anterior muscle. To better demonstrate the role of glucocorticoids in the response of GS to fasting, we suppressed their action by RU38486 administration (a potent glucocorticoid antagonist) and their production by adrenalectomy in fed and fasted rats. An increase in fasting-induced GS activity was observed in skeletal muscles from both adult and aged rats. Adrenalectomy, but surprisingly not RU38486, suppressed the fasting-induced increase in GS activity and expression. The data clearly show that the GS responsiveness to fasting was not modified by aging in skeletal muscle.

Urea application promotes amino acid metabolism and membrane lipid peroxidation in Azolla.

PubMed

Chen, Jiana; Huang, Min; Cao, Fangbo; Pardha-Saradhi, P; Zou, Yingbin

2017-01-01

A pot experiment was conducted to evaluate the effect of urea on nitrogen metabolism and membrane lipid peroxidation in Azolla pinnata. Compared to controls, the application of urea to A. pinnata resulted in a 44% decrease in nitrogenase activity, no significant change in glutamine synthetase activity, 660% higher glutamic-pyruvic transaminase, 39% increase in free amino acid levels, 22% increase in malondialdehyde levels, 21% increase in Na+/K+- levels, 16% increase in Ca2+/Mg2+-ATPase levels, and 11% decrease in superoxide dismutase activity. In terms of H2O2 detoxifying enzymes, peroxidase activity did not change and catalase activity increased by 64% in urea-treated A. pinnata. These findings suggest that urea application promotes amino acid metabolism and membrane lipid peroxidation in A. pinnata.

The action of Saraca asoca Roxb. de Wilde bark on the PGH2 synthetase enzyme complex of the sheep vesicular gland.

PubMed

Middelkoop, T B; Labadie, R P

1985-01-01

Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH2 synthetase. They were assayed spectrophotometrically with adrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.

Yeast Mitochondrial Leucyl-tRNA Synthetase CP1 Domain Has Functionally Diverged to Accommodate RNA Splicing at Expense of Hydrolytic Editing*

PubMed Central

Sarkar, Jaya; Poruri, Kiranmai; Boniecki, Michal T.; McTavish, Katherine K.; Martinis, Susan A.

2012-01-01

The yeast mitochondrial leucyl-tRNA synthetase (ymLeuRS) performs dual essential roles in group I intron splicing and protein synthesis. A specific LeuRS domain called CP1 is responsible for clearing noncognate amino acids that are misactivated during aminoacylation. The ymLeuRS CP1 domain also plays a critical role in splicing. Herein, the ymLeuRS CP1 domain was isolated from the full-length enzyme and was active in RNA splicing in vitro. Unlike its Escherichia coli LeuRS CP1 domain counterpart, it failed to significantly hydrolyze misaminoacylated tRNALeu. In addition and in stark contrast to the yeast domain, the editing-active E. coli LeuRS CP1 domain failed to recapitulate the splicing activity of the full-length E. coli enzyme. Although LeuRS-dependent splicing activity is rooted in an ancient adaptation for its aminoacylation activity, these results suggest that the ymLeuRS has functionally diverged to confer a robust splicing activity. This adaptation could have come at some expense to the protein's housekeeping role in aminoacylation and editing. PMID:22383526

Amino acid metabolism in maize earshoots. Implications for assimilate preconditioning and nitrogen signaling.

PubMed

Seebauer, Juliann R; Moose, Stephen P; Fabbri, Bradon J; Crossland, Lyle D; Below, Frederick E

2004-12-01

Nitrogen (N) is an essential requirement for kernel growth in maize (Zea mays); however, little is known about how N assimilates are metabolized in young earshoots during seed development. The objective of this study was to assess amino acid metabolism in cob and spikelet tissues during the critical 2 weeks following silking. Two maize hybrids were grown in the field for 2 years at two levels of supplemental N fertilizer (0 and 168 kg N/ha). The effects of the reproductive sink on cob N metabolism were examined by comparing pollinated to unpollinated earshoots. Earshoots were sampled at 2, 8, 14, and 18 d after silking; dissected into cob, spikelet, and/or pedicel and kernel fractions; then analyzed for amino acid profiles and key enzyme activities associated with amino acid metabolism. Major amino acids in the cob were glutamine (Gln), aspartic acid (Asp), asparagine (Asn), glutamate, and alanine. Gln concentrations dropped dramatically from 2 to 14 d after silking in both pollinated and unpollinated cobs, whereas all other measured amino acids accumulated over time in unpollinated spikelets and cobs, especially Asn. N supply had a variable effect on individual amino acid levels in young cobs and spikelets, with Asn being the most notably enhanced. We found that the cob performs significant enzymatic interconversions among Gln, alanine, Asp, and Asn during early reproductive development, which may precondition the N assimilate supply for sustained kernel growth. The measured amino acid profiles and enzymatic activities suggest that the Asn to Gln ratio in cobs may be part of a signal transduction pathway involving aspartate aminotransferase, Gln synthetase, and Asn synthetase to indicate plant N status for kernel development.

The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡

PubMed Central

Shah, Manish B.; Ingram-Smith, Cheryl; Cooper, Leroy L.; Qu, Jun; Meng, Yu; Smith, Kerry S.; Gulick, Andrew M.

2009-01-01

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket. PMID:19544569

Monoclonal Antibodies Against Tyrosyl-tRNA Synthetase and Its Isolated Cytokine-Like Domain

PubMed Central

Khoruzenko, Antonina; Cherednyk, Olga; Filonenko, Valeriy; Kornelyuk, Aleksander

2013-01-01

Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH2-terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXC-chemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N- and C-terminal domains of TyrRS. Recombinant TyrRS and its N- and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization. PMID:23750478

Incorporation of Extracellular Fatty Acids by a Fatty Acid Kinase-Dependent Pathway in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Frank, Matthew W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Summary Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids. PMID:24673884

Natural toxins that affect plant amino acid metabolism

USDA-ARS?s Scientific Manuscript database

A diverse range of natural compounds interfere with the synthesis and other aspects of amino acid metabolism. Some are amino acid analogues, but most are not. This review covers a number of specific natural phytotoxic compounds by molecular target site. Inhibition of glutamine synthetase is of part...

Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype

PubMed Central

2013-01-01

Background Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. Methods We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. Results We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. Conclusion This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes. PMID:24103465

Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

PubMed

Tzagoloff, A; Shtanko, A

1995-06-01

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Membrane anchoring of aminoacyl-tRNA synthetases by convergent acquisition of a novel protein domain.

PubMed

Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A G; Ribas de Pouplana, Lluis; Luque, Ignacio

2011-11-25

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.

Methods for Kinetic and Thermodynamic Analysis of Aminoacyl-tRNA Synthetases

PubMed Central

Francklyn, Christopher S.; First, Eric A.; Perona, John J.; Hou, Ya-Ming

2008-01-01

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion. PMID:18241792

Urea application promotes amino acid metabolism and membrane lipid peroxidation in Azolla

PubMed Central

Chen, Jiana; Cao, Fangbo; Pardha-Saradhi, P.; Zou, Yingbin

2017-01-01

A pot experiment was conducted to evaluate the effect of urea on nitrogen metabolism and membrane lipid peroxidation in Azolla pinnata. Compared to controls, the application of urea to A. pinnata resulted in a 44% decrease in nitrogenase activity, no significant change in glutamine synthetase activity, 660% higher glutamic-pyruvic transaminase, 39% increase in free amino acid levels, 22% increase in malondialdehyde levels, 21% increase in Na+/K+- levels, 16% increase in Ca2+/Mg2+-ATPase levels, and 11% decrease in superoxide dismutase activity. In terms of H2O2 detoxifying enzymes, peroxidase activity did not change and catalase activity increased by 64% in urea-treated A. pinnata. These findings suggest that urea application promotes amino acid metabolism and membrane lipid peroxidation in A. pinnata. PMID:28945775

Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

PubMed

Brown, J R; Doolittle, W F

1995-03-28

Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an

Identification of protein interfaces within the multi-aminoacyl-tRNA synthetase complex: the case of lysyl-tRNA synthetase and the scaffold protein p38.

PubMed

Rémion, Azaria; Khoder-Agha, Fawzi; Cornu, David; Argentini, Manuela; Redeker, Virginie; Mirande, Marc

2016-07-01

Human cytoplasmic lysyl-tRNA synthetase (LysRS) is associated within a multi-aminoacyl-tRNA synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC-associated and MSC-dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N-terminal peptide of p38 was reported [Ofir-Birin Y et al. (2013) Mol Cell 49, 30-42]. In order to identify amino acid residues involved in interaction of the two proteins, the non-natural, photo-cross-linkable amino acid p-benzoyl-l-phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross-linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross-linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.

Inhibition of Arabidopsis growth by the allelopathic compound azetidine-2-carboxylate is due to the low amino acid specificity of cytosolic prolyl-tRNA synthetase.

PubMed

Lee, Jiyeon; Joshi, Naveen; Pasini, Rita; Dobson, Renwick C J; Allison, Jane; Leustek, Thomas

2016-10-01

The toxicity of azetidine-2-carboxylic acid (A2C), a structural analogue of L-proline, results from its incorporation into proteins due to misrecognition by prolyl-tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS-Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS-Cyt (At3g62120) is cytosolic. AtProRS-Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His-tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP-PPi exchange and aminoacylation assays, and showed similar K m for L-proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS-Cyt showed nearly identical substrate specificity for L-proline and A2C, but for AtProRS-Org the specificity constant was 77.6 times higher for L-proline than A2C, suggesting that A2C-sensitivity may result from lower amino acid specificity of AtProRS-Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

PubMed

Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

2016-05-01

Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

Evolutionary anomalies among the aminoacyl-tRNA synthetases

NASA Technical Reports Server (NTRS)

Doolittle, R. F.; Handy, J.; Bada, J. L. (Principal Investigator)

1998-01-01

Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises - is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.

Compositions of orthogonal glutamyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

DOEpatents

Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA; Santoro, Stephen [Cambridge, MA

2009-05-05

Compositions and methods of producing components of protein biosynthetic machinery that include glutamyl orthogonal tRNAs, glutamyl orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of glutamyl tRNAs/synthetases are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins using these orthogonal pairs.

The Cytoplasmic Prolyl-tRNA Synthetase of the Malaria Parasite is a Dual-Stage Target for Drug Development

PubMed Central

Herman, Jonathan D.; Pepper, Lauren R.; Cortese, Joseph F.; Estiu, Guillermina; Galinsky, Kevin; Zuzarte-Luis, Vanessa; Derbyshire, Emily R.; Ribacke, Ulf; Lukens, Amanda K.; Santos, Sofia A.; Patel, Vishal; Clish, Clary B.; Sullivan, William J.; Zhou, Huihao; Bopp, Selina E.; Schimmel, Paul; Lindquist, Susan; Clardy, Jon; Mota, Maria M.; Keller, Tracy L.; Whitman, Malcolm; Wiest, Olaf; Wirth, Dyann F.; Mazitschek, Ralph

2015-01-01

The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for the development of the next-generation of antimalarial drugs. Using an integrated chemogenomics approach that combined drug-resistance selection, whole genome sequencing and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivatives such as halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the P. berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is highly active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses, and represents a promising lead for the development of dual-stage next generation antimalarials. PMID:25995223

Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

PubMed Central

Pei, Zhengtong; Sun, Peng; Huang, Ping; Lal, Bachchu; Laterra, John; Watkins, Paul A.

2009-01-01

The contribution of lipid metabolic pathways to malignancy is poorly understood. Expression of the fatty acyl-CoA synthetase, ACSVL3, was found to be markedly elevated in clinical malignant glioma specimens but nearly undetectable in normal glia. ACSVL3 levels correlated with the malignant behavior of human glioma cell lines and glioma cells propagated as xenografts. ACSVL3 expression was induced by the activation of oncogenic receptor tyrosine kinases (RTK) c-Met and EGFR. Inhibiting c-Met activation with neutralizing anti-HGF monoclonal antibodies reduced ACSVL3 expression concurrent with tumor growth inhibition in vivo. ACSVL3 expression knockdown using RNA interference, which decreased long-chain fatty acid activation, inhibited anchorage-dependent and anchorage-independent glioma cell growth by ~70% and ~ 90%, respectively. ACSVL3-depleted cells were less tumorigenic than control cells and subcutaneous xenografts grew ~60% slower than control tumors. Orthotopic xenografts produced by ACSVL3-depleted cells were 82–86 % smaller than control xenografts. ACSVL3 knockdown disrupted Akt function as evidenced by RTK-induced transient decreases in total and phosphorylated Akt, as well as GSK3β, via a caspase-dependent mechanism. Expressing constitutively active myr-Akt rescued cells from the anchorage-dependent and anchorage-independent growth inhibitory effects of ACSVL3 depletion. These studies show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function. PMID:19920185

Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

PubMed

Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

2016-08-01

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

Henoch-Schönlein purpura nephritis occurring postpartum in a patient with anti-PL-7 anti-synthetase syndrome.

PubMed

Nagai, Kojiro; Kishi, Jun; Morizumi, Shun; Minakuchi, Jun; Bando, Yoshimi; Nishioka, Yasuhiko; Doi, Toshio

2017-09-01

A 37-year-old pregnant woman developed purpura which was subsequently diagnosed as Henoch-Schönlein purpura (HSP). After childbirth, the patient developed proteinuria and hematuria. Further examination revealed that the HSP nephritis (HSPN) was associated with anti-threonyl-tRNA synthetase anti-synthetase syndrome. The onset of HSPN during pregnancy or after childbirth is rare. Moreover, to our knowledge, this is the first case to describe renal involvement in anti-synthetase syndrome.

New Aminoacyl-tRNA Synthetase-like Protein in Insecta with an Essential Mitochondrial Function*♦

PubMed Central

Guitart, Tanit; Leon Bernardo, Teresa; Sagalés, Jessica; Stratmann, Thomas; Bernués, Jordi; Ribas de Pouplana, Lluís

2010-01-01

Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria. PMID:20870726

Engineering posttranslational proofreading to discriminate nonstandard amino acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunjapur, Aditya M.; Stork, Devon A.; Kuru, Erkin

Accurate incorporation of nonstandard amino acids (nsAAs) is central for genetic code expansion to increase the chemical diversity of proteins. However, aminoacyl-tRNA synthetases are polyspecific and facilitate incorporation of multiple nsAAs. We investigated and repurposed a natural protein degradation pathway, the N-end rule pathway, to devise an innovative system for rapid assessment of the accuracy of nsAA incorporation. Using this tool to monitor incorporation of the nsAA biphenylalanine allowed the identification of tyrosyl-tRNA synthetase (TyrRS) variants with improved amino acid specificity. The evolved TyrRS variants enhanced our ability to contain unwanted proliferation of genetically modified organisms. In conclusion, this posttranslationalmore » proofreading system will aid the evolution of orthogonal translation systems for specific incorporation of diverse nsAAs.« less

Engineering posttranslational proofreading to discriminate nonstandard amino acids

DOE PAGES

Kunjapur, Aditya M.; Stork, Devon A.; Kuru, Erkin; ...

2018-01-04

Accurate incorporation of nonstandard amino acids (nsAAs) is central for genetic code expansion to increase the chemical diversity of proteins. However, aminoacyl-tRNA synthetases are polyspecific and facilitate incorporation of multiple nsAAs. We investigated and repurposed a natural protein degradation pathway, the N-end rule pathway, to devise an innovative system for rapid assessment of the accuracy of nsAA incorporation. Using this tool to monitor incorporation of the nsAA biphenylalanine allowed the identification of tyrosyl-tRNA synthetase (TyrRS) variants with improved amino acid specificity. The evolved TyrRS variants enhanced our ability to contain unwanted proliferation of genetically modified organisms. In conclusion, this posttranslationalmore » proofreading system will aid the evolution of orthogonal translation systems for specific incorporation of diverse nsAAs.« less

Discovery of antimicrobial compounds targeting bacterial type FAD synthetases.

PubMed

Sebastián, María; Anoz-Carbonell, Ernesto; Gracia, Begoña; Cossio, Pilar; Aínsa, José Antonio; Lans, Isaías; Medina, Milagros

2018-12-01

The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammoniagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also evaluated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

PubMed

Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

2016-08-02

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33

PubMed Central

Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

2016-01-01

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F1-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata. PMID:27490569

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

PubMed Central

Iñón de Iannino, Nora; Briones, Gabriel; Tolmasky, Marcelo; Ugalde, Rodolfo A.

1998-01-01

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in Brucella infection. PMID:9721274

Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

PubMed

Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

2014-10-01

Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

PubMed Central

Gosling, J. P.; Duggan, P. F.

1971-01-01

Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle. PMID:5557595

Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor

PubMed Central

Krall, Abigail S.; Xu, Shili; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

2016-01-01

Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation. PMID:27126896

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brizio, Carmen; Galluccio, Michele; Wait, Robin

2006-06-09

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-stepmore » affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.« less

Protein synthesis editing by a DNA aptamer.

PubMed Central

Hale, S P; Schimmel, P

1996-01-01

Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response. Images Fig. 3 Fig. 4 PMID:8610114

Abscisic Acid Regulates Inflammation via Ligand-binding Domain-independent Activation of Peroxisome Proliferator-activated Receptor γ*

PubMed Central

Bassaganya-Riera, Josep; Guri, Amir J.; Lu, Pinyi; Climent, Montse; Carbo, Adria; Sobral, Bruno W.; Horne, William T.; Lewis, Stephanie N.; Bevan, David R.; Hontecillas, Raquel

2011-01-01

Abscisic acid (ABA) has shown efficacy in the treatment of diabetes and inflammation; however, its molecular targets and the mechanisms of action underlying its immunomodulatory effects remain unclear. This study investigates the role of peroxisome proliferator-activated receptor γ (PPAR γ) and lanthionine synthetase C-like 2 (LANCL2) as molecular targets for ABA. We demonstrate that ABA increases PPAR γ reporter activity in RAW 264.7 macrophages and increases ppar γ expression in vivo, although it does not bind to the ligand-binding domain of PPAR γ. LANCL2 knockdown studies provide evidence that ABA-mediated activation of macrophage PPAR γ is dependent on lancl2 expression. Consistent with the association of LANCL2 with G proteins, we provide evidence that ABA increases cAMP accumulation in immune cells. ABA suppresses LPS-induced prostaglandin E2 and MCP-1 production via a PPAR γ-dependent mechanism possibly involving activation of PPAR γ and suppression of NF-κB and nuclear factor of activated T cells. LPS challenge studies in PPAR γ-expressing and immune cell-specific PPAR γ null mice demonstrate that ABA down-regulates toll-like receptor 4 expression in macrophages and T cells in vivo through a PPAR γ-dependent mechanism. Global transcriptomic profiling and confirmatory quantitative RT-PCR suggest novel candidate targets and demonstrate that ABA treatment mitigates the effect of LPS on the expression of genes involved in inflammation, metabolism, and cell signaling, in part, through PPAR γ. In conclusion, ABA decreases LPS-mediated inflammation and regulates innate immune responses through a bifurcating pathway involving LANCL2 and an alternative, ligand-binding domain-independent mechanism of PPAR γ activation. PMID:21088297

Abscisic acid regulates inflammation via ligand-binding domain-independent activation of peroxisome proliferator-activated receptor gamma.

PubMed

Bassaganya-Riera, Josep; Guri, Amir J; Lu, Pinyi; Climent, Montse; Carbo, Adria; Sobral, Bruno W; Horne, William T; Lewis, Stephanie N; Bevan, David R; Hontecillas, Raquel

2011-01-28

Abscisic acid (ABA) has shown efficacy in the treatment of diabetes and inflammation; however, its molecular targets and the mechanisms of action underlying its immunomodulatory effects remain unclear. This study investigates the role of peroxisome proliferator-activated receptor γ (PPAR γ) and lanthionine synthetase C-like 2 (LANCL2) as molecular targets for ABA. We demonstrate that ABA increases PPAR γ reporter activity in RAW 264.7 macrophages and increases ppar γ expression in vivo, although it does not bind to the ligand-binding domain of PPAR γ. LANCL2 knockdown studies provide evidence that ABA-mediated activation of macrophage PPAR γ is dependent on lancl2 expression. Consistent with the association of LANCL2 with G proteins, we provide evidence that ABA increases cAMP accumulation in immune cells. ABA suppresses LPS-induced prostaglandin E(2) and MCP-1 production via a PPAR γ-dependent mechanism possibly involving activation of PPAR γ and suppression of NF-κB and nuclear factor of activated T cells. LPS challenge studies in PPAR γ-expressing and immune cell-specific PPAR γ null mice demonstrate that ABA down-regulates toll-like receptor 4 expression in macrophages and T cells in vivo through a PPAR γ-dependent mechanism. Global transcriptomic profiling and confirmatory quantitative RT-PCR suggest novel candidate targets and demonstrate that ABA treatment mitigates the effect of LPS on the expression of genes involved in inflammation, metabolism, and cell signaling, in part, through PPAR γ. In conclusion, ABA decreases LPS-mediated inflammation and regulates innate immune responses through a bifurcating pathway involving LANCL2 and an alternative, ligand-binding domain-independent mechanism of PPAR γ activation.

The inhibition of nitric oxide-activated poly(ADP-ribose) synthetase attenuates transsynaptic alteration of spinal cord dorsal horn neurons and neuropathic pain in the rat.

PubMed

Mao, J; Price, D D; Zhu, J; Lu, J; Mayer, D J

1997-09-01

Transsynaptic alteration of spinal cord dorsal horn neurons characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called 'dark' neurons) occurs in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the common sciatic nerve. The incidence of dark neurons in CCI rats has been proposed to be mediated by glutamate-induced neurotoxicity. In the present study, we examined whether the inhibition of the nitric oxide (NO)-activated poly(ADP-ribose) synthetase (PARS), a nuclear enzyme critical to glutamate-induced neurotoxicity, would both reduce the incidence of dark neurons and attenuate behavioral manifestations of neuropathic pain in CCI rats. Dark neurons were observed bilaterally (with ipsilateral predominance) within the spinal cord dorsal horn, particularly in laminae I-II, of rats 8 days after unilateral sciatic nerve ligation as compared to sham operated rats. The number of dark neurons in the dorsal horn was dose-dependently reduced in CCI rats receiving once daily intrathecal (i.t.) treatment with the PARS inhibitor benzamide (200 or 400 nmol, but not 100 nmol benzamide or saline) for 7 days. Consistent with the histological improvement, thermal hyperalgesia, mechanical hyperalgesia, and low threshold mechano-allodynia also were reliably reduced in CCI rats treated with either 200 or 400 nmol benzamide. Neither dark neurons nor neuropathic pain behaviors were reliably affected by i.t. administration of either 800 nmol novobiocin (a mono(ADP-ribose) synthetase) or 800 nmol benzoic acid (the backbone structure of benzamide), indicating a selective effect of benzamide. Intrathecal treatment with an NO synthase inhibitor NG-nitro-L-arginine methyl ester (40 nmol, but not its inactive D-isomer) utilizing the same benzamide treatment regimen resulted in similar reductions of both dark neurons and neuropathic pain behaviors in CCI rats. These results provide, for the first time, in vivo evidence indicating that benzamide is

Unique regulatory properties of the UDP-glucose:. beta. -1,4-glucan synthetase of Acetobacter xylinum. [Acetobacter xylinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benziman, M.; Aloni, Y.; Delmer, D.P.

1983-01-01

Conditions have been found for an extremely efficient transfer of glucose from UDP-glucose to a cellulosic ..beta..-1,4-glucan product, using enzyme preparations derived from cells of Acetobacter xylinum. Membrane fractions obtained by rupturing cells in the presence of 20% (w/v) polyethylene glycol-4000 (PEG-4000) exhibited UDP-glucose:..beta..-1,4-glucan synthetase activity 3- to 10-fold higher than those previously reported. Enzyme prepared in this fashion also shows a further marked activation by GTP. The activation (apparent K/sub alpha/ = 35 ..mu..M) is quite specific for GTP. A variety of other nucleotides and nucleotide derivatives had no effect on activity. Guanosine-5'-(lambda-thio)triphosphate, an analog of GTP, is evenmore » more efficient than GTP (K/sub alpha/ = 17 ..mu..M). Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor essential for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself is devoid of synthetase activity and does not stimulate activity of the enzyme in the absence of GTP. Under optimal conditions, in the presence of GTP, factor, and PEG-4000, initial rates of enzyme activity that are 200 times higher than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose. 26 references, 3 figures, 3 tables.« less

Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

PubMed

Teodoro, Bruno G; Sampaio, Igor H; Bomfim, Lucas H M; Queiroz, André L; Silveira, Leonardo R; Souza, Anderson O; Fernandes, Anna M A P; Eberlin, Marcos N; Huang, Tai-Yu; Zheng, Donghai; Neufer, P Darrell; Cortright, Ronald N; Alberici, Luciane C

2017-02-01

Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or β-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

PubMed Central

Gebhardt, R; Mecke, D

1983-01-01

The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells. Images Fig. 1. PMID:6138251

Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors.

PubMed

Rode, W; Jastreboff, M M

1984-01-01

Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.

Co-operation between Polymerases and Nucleotide Synthetases in the RNA World.

PubMed

Kim, Ye Eun; Higgs, Paul G

2016-11-01

It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes). The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates), and non-functional mutants of the two sequences (which act as parasites). Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.

Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

DOEpatents

Anderson, J Christopher [San Francisco, CA; Wu, Ning [Brookline, MA; Santoro, Stephen [Cambridge, MA; Schultz, Peter G [La Jolla, CA

2009-12-29

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

DOEpatents

Anderson, J Christopher [San Francisco, CA; Wu, Ning [Brookline, MA; Santoro, Stephen [Cambridge, MA; Schultz, Peter G [La Jolla, CA

2011-10-04

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

DOEpatents

Anderson, J Christopher [San Francisco, CA; Wu, Ning [Brookline, MA; Santoro, Stephen [Cambridge, MA; Schultz, Peter G [La Jolla, CA

2009-08-18

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

Combinatorial assembly of simple and complex D-lysergic acid alkaloid peptide classes in the ergot fungus Claviceps purpurea.

PubMed

Ortel, Ingo; Keller, Ullrich

2009-03-13

The ergot fungus Claviceps purpurea produces both ergopeptines and simple d-lysergic acid alkylamides. In the ergopeptines, such as ergotamine, d-lysergic acid is linked to a bicyclic tripeptide in amide-like fashion, whereas in the d-lysergylalkanolamides it is linked to an amino alcohol derived from alanine. We show here that these compound classes are synthesized by a set of three non-ribosomal lysergyl peptide synthetases (LPSs), which interact in a combinatorial fashion for synthesis of the relevant product. The trimodular LPS1 assembles with LPS2, the d-lysergic acid recruiting module, to synthesize the d-lysergyltripeptide precursors of ergopeptines from d-lysergic acid and the three amino acids of the peptide chain. Alternatively, LPS2 can assemble with a distinct monomodular non-ribosomal peptide synthetase (NRPS) subunit (ergometrine synthetase) to synthesize the d-lysergic acid alkanolamide ergometrine from d-lysergic acid and alanine. The synthesis proceeds via covalently bound d-lysergyl alanine and release of dipeptide as alcohol with consumption of NADPH. Enzymatic and immunochemical analyses showed that ergometrine synthetase is most probably the enzyme LPS3 whose gene had been identified previously as part of the ergot alkaloid biosynthesis gene cluster in C. purpurea. Inspections of all LPS sequences showed no recognizable peptide linkers for their protein-protein interactions as in NRPS subunits of bacteria. Instead, they all carry conserved N-terminal domains (C0-domains) with similarity to the C-terminal halves of NRPS condensation domains pointing to an alternative mechanism of subunit-subunit interactions in fungal NRPS systems. Phylogenetic analysis of LPS modules and the C0-domains suggests that these enzyme systems most probably evolved by module duplications and rearrangements from a bimodular ancestor.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman

2007-02-01

DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K{sub 2}) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetricmore » unit.« less

Editing activity for eliminating mischarged tRNAs is essential in mammalian mitochondria

PubMed Central

Hilander, Taru; Zhou, Xiao-Long; Konovalova, Svetlana; Zhang, Fu-Ping; Euro, Liliya; Chilov, Dmitri; Poutanen, Matti; Chihade, Joseph

2018-01-01

Abstract Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms. PMID:29228266

Structure-function studies of adenylosuccinate synthetase from Escherichia coli.

PubMed

Honzatko, R B; Fromm, H J

1999-10-01

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, thermodynamically coupling the hydrolysis of GTP to the formation of adenylosuccinate from l-aspartate and IMP. The enzyme from Esherichia coli undergoes a ligand-induced dimerization, which leads to the assembly of a complete active site. The binding of IMP causes conformational changes over distances of 30 A, the end result of which is the activation of essential catalytic elements and the organization of the binding pocket for Mg(2+)-GTP. The enzyme promotes first a phosphoryl transfer from GTP to the 6-oxygen atom of IMP, by way of a transition state that has characteristics of both associative and dissociative reaction pathways. Following the formation of 6-phosphoryl-IMP, the enzyme then catalyzes the nucleophilic displacement of the 6-phosphoryl group by the alpha-amino group of l-aspartate in a transition state, which requires two metal cations. Copyright 1999 Academic Press.

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

PubMed Central

Mofford, David M.; Reddy, Gadarla Randheer; Miller, Stephen C.

2014-01-01

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation. PMID:24616520

Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.

PubMed

Khan, Sameena; Garg, Ankur; Camacho, Noelia; Van Rooyen, Jason; Kumar Pole, Anil; Belrhali, Hassan; Ribas de Pouplana, Lluis; Sharma, Vinay; Sharma, Amit

2013-05-01

Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively.

Combinatorial Assembly of Simple and Complex d-Lysergic Acid Alkaloid Peptide Classes in the Ergot Fungus Claviceps purpurea*S⃞

PubMed Central

Ortel, Ingo; Keller, Ullrich

2009-01-01

The ergot fungus Claviceps purpurea produces both ergopeptines and simple d-lysergic acid alkylamides. In the ergopeptines, such as ergotamine, d-lysergic acid is linked to a bicyclic tripeptide in amide-like fashion, whereas in the d-lysergylalkanolamides it is linked to an amino alcohol derived from alanine. We show here that these compound classes are synthesized by a set of three non-ribosomal lysergyl peptide synthetases (LPSs), which interact in a combinatorial fashion for synthesis of the relevant product. The trimodular LPS1 assembles with LPS2, the d-lysergic acid recruiting module, to synthesize the d-lysergyltripeptide precursors of ergopeptines from d-lysergic acid and the three amino acids of the peptide chain. Alternatively, LPS2 can assemble with a distinct monomodular non-ribosomal peptide synthetase (NRPS) subunit (ergometrine synthetase) to synthesize the d-lysergic acid alkanolamide ergometrine from d-lysergic acid and alanine. The synthesis proceeds via covalently bound d-lysergyl alanine and release of dipeptide as alcohol with consumption of NADPH. Enzymatic and immunochemical analyses showed that ergometrine synthetase is most probably the enzyme LPS3 whose gene had been identified previously as part of the ergot alkaloid biosynthesis gene cluster in C. purpurea. Inspections of all LPS sequences showed no recognizable peptide linkers for their protein-protein interactions as in NRPS subunits of bacteria. Instead, they all carry conserved N-terminal domains (C0-domains) with similarity to the C-terminal halves of NRPS condensation domains pointing to an alternative mechanism of subunit-subunit interactions in fungal NRPS systems. Phylogenetic analysis of LPS modules and the C0-domains suggests that these enzyme systems most probably evolved by module duplications and rearrangements from a bimodular ancestor. PMID:19139103

The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel

The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of cropmore » yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.« less

The toxic effects of diethyl phthalate on the activity of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

PubMed

Cheng, Tai-Sheng

2012-11-15

The toxic effects of diethyl phthalate (DEP), a potent allelochemical, on the enzyme activity and polypeptide accumulation of glutamine synthetase (GS) in greater duckweed were investigated. In our previous studies, DEP induced oxidative responses at concentrations from 0.5 to 2 mM in greater duckweed and the antioxidant enzymes played important roles in the defense strategy against DEP stress. In this study, DAB-H(2)O(2) and NBT stain for superoxide radicals (O(2)(·-)), lipid peroxidation, HSP70, and ammonia accumulation in DEP-treated duckweed tissues revealed adverse effect of DEP in plant growth. Biochemical analysis and physiological methods were combined to investigate GS activity and polypeptide accumulation under DEP-induced stress. The results showed that GS activity was reduced with the increasing concentration of DEP, indicative of enhanced toxic effect. Immunoblot analysis with chloroplast soluble fractions indicated that the chloroplastic GS (GS2) polypeptide from greater duckweed was degraded under DEP stress conditions. The response of GS2 to the DEP stress may be modulated by means of redox change in plant tissues, chloroplasts, and chloroplast lysates. The results suggest that DEP is toxic to the greater duckweed by inhibition of the GS isoenzymes in nitrogen assimilation and the GS2 plays important roles in the adaptation strategy against DEP toxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

PubMed

Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je; Choi, Yoon Seo; Chung, Jeong Min; Lee, Sangmin; Kim, Hoi Kyoung; Kim, Jong Hyun; Eom, Chi-Yong; Kim, Yeon-Gil; Guo, Min; Jung, Hyun Suk; Kang, Beom Sik; Kim, Sunghoon

2015-12-04

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

PubMed Central

Chadha, Sanya; Mallampudi, N. Arjunreddy; Mohapatra, Debendra K.

2017-01-01

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

PubMed

Chadha, Sanya; Mallampudi, N Arjunreddy; Mohapatra, Debendra K; Madhubala, Rentala

2017-01-01

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase ( Ld LysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. Ld LysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas Ld LysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized Ld LysRS-1. Recombinant Ld LysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The Ld LysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. Ld LysRS-1 appears to be an essential gene, as a chromosomal knockout of Ld LysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC 50 ], 4.19 µM) and intracellular amastigotes (IC 50 , 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using Ld LysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant Ld LysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random-coil-like pKa values.

PubMed

Cozza, Concetta; Neira, José L; Florencio, Francisco J; Muro-Pastor, M Isabel; Rizzuti, Bruno

2017-06-01

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK a values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK a values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7. © 2017 The Protein Society.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

PubMed Central

Guerreiro, Juliano R.; Lameu, Claudiana; Oliveira, Eduardo F.; Klitzke, Clécio F.; Melo, Robson L.; Linares, Edlaine; Augusto, Ohara; Fox, Jay W.; Lebrun, Ivo; Serrano, Solange M. T.; Camargo, Antonio C. M.

2009-01-01

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases. PMID:19491403

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

PubMed

Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

2010-07-01

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

Chronic exposure to glufosinate-ammonium induces spatial memory impairments, hippocampal MRI modifications and glutamine synthetase activation in mice.

PubMed

Calas, André-Guilhem; Richard, Olivier; Même, Sandra; Beloeil, Jean-Claude; Doan, Bich-Thuy; Gefflaut, Thierry; Même, William; Crusio, Wim E; Pichon, Jacques; Montécot, Céline

2008-07-01

Glufosinate-ammonium (GLA), the active compound of a worldwide-used herbicide, acts by inhibiting the plant glutamine synthetase (GS) leading to a lethal accumulation of ammonia. GS plays a pivotal role in the mammalian brain where it allows neurotransmitter glutamate recycling within astroglia. Clinical studies report that an acute GLA ingestion induces convulsions and memory impairment in humans. Toxicological studies performed at doses used for herbicidal activity showed that GLA is probably harmless at short or medium range periods. However, effects of low doses of GLA on chronically exposed subjects are not known. In our study, C57BL/6J mice were treated during 10 weeks three times a week with 2.5, 5 and 10mg/kg of GLA. Effects of this chronic treatment were assessed at behavioral, structural and metabolic levels by using tests of spatial memory, locomotor activity and anxiety, hippocampal magnetic resonance imaging (MRI) texture analysis, and hippocampal GS activity assay, respectively. Chronic GLA treatments have effects neither on anxiety nor on locomotor activity of mice but at 5 and 10mg/kg induce (1) mild memory impairments, (2) a modification of hippocampal texture and (3) a significant increase in hippocampal GS activity. It is suggested that these modifications may be causally linked one to another. Since glutamate is the main neurotransmitter in hippocampus where it plays a crucial role in spatial memory, hippocampal MRI texture and spatial memory alterations might be the consequences of hippocampal glutamate homeostasis modification revealed by increased GS activity in hippocampus. The present study provides the first data that show cerebral alterations after chronic exposure to GLA.

S-Adenosylmethionine synthetase 3 is important for pollen tube growth

USDA-ARS?s Scientific Manuscript database

S-Adenosylmethionine is widely used in a variety of biological reactions and participates in the methionine (Met) metabolic pathway. In Arabidopsis (Arabidopsis thaliana), one of the four S-adenosylmethionine synthetase genes, METHIONINE ADENOSYLTRANSFERASE3 (MAT3), is highly expressed in pollen. He...

Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta.

PubMed Central

Kent, R S; Diedrich, S L; Whorton, A R

1983-01-01

To address the hypothesis that metabolites of arachidonic acid are important regulators of prostaglandin (PG) synthesis in intact vascular tissue, we studied arachidonate metabolism in rabbit aortas in response to a continuous infusion of arachidonic acid, 10 micrograms/ml. Prostacyclin (PGI2; measured as 6-keto-PGF1 alpha) production rate accelerated during the first 2 min, reached peak velocity at 2 min, and then progressively decelerated. The velocity profile of PGI2 production was similar to that previously reported for cyclooxygenase holoenzyme assayed in vitro, and was consistent with progressive inactivation of the enzymes leading to PGI2 synthesis. We determined the specific inhibition of cyclooxygenase and prostacyclin synthetase by measuring PGI2 and PGE2 production rates and by infusing cyclic endoperoxides. Our results indicate preferential inactivation of cyclooxygenase during arachidonate metabolism, most likely due to cyclooxygenase-derived oxidative intermediates. This was a dose-dependent response and resulted in a progressive decrease in the 6-keto-PGF1 alpha/PGE2 ratio. Exogenously added 15-hydroperoxy eicosatetraenoic acid, on the other hand, actually stimulated cyclooxygenase activity at low doses, while markedly inhibiting prostacyclin synthetase. This finding, along with the accelerating nature of arachidonate metabolism, is consistent with the concept of "peroxide tone" as a mediator of cyclooxygenase activity in this system. These results demonstrate that arachidonate metabolites regulate PG synthesis in intact blood vessels. The progressive enzymatic inhibition intrinsic to arachidonate metabolism may be a model for similar changes occurring in states of enhanced lipid peroxidation. These metabolic alterations might greatly influence the numerous vascular functions known to involve arachidonic acid metabolism. PMID:6409932

Assessing the effects of threonyl-tRNA synthetase on angiogenesis-related responses.

PubMed

Mirando, Adam C; Abdi, Khadar; Wo, Peibin; Lounsbury, Karen M

2017-01-15

Several recent reports have found a connection between specific aminoacyl-tRNA synthetases and the regulation of angiogenesis. As this new area of research is explored, it is important to have reliable assays to assess the specific angiogenesis functions of these enzymes. This review provides information about specific in vitro and in vivo methods that were used to assess the angiogenic functions of threonyl-tRNA synthetase including endothelial cell migration and tube assays as well as chorioallantoic membrane and tumor vascularization assays. The theory and discussion include best methods of analysis and quantification along with the advantages and limitations of each type of assay. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of glutamine synthetase II activity in Rhizobium meliloti 104A14.

PubMed Central

Shatters, R G; Somerville, J E; Kahn, M L

1989-01-01

Most rhizobia contain two glutamine synthetase (GS) enzymes: GSI, encoded by glnA, and GSII, encoded by glnII. We have found that WSU414, a Rhizobium meliloti 104A14 glutamine auxotroph derived from a glnA parental strain, is an ntrA mutant. The R. meliloti glnII promoter region contains DNA sequences similar to those found in front of other genes that require ntrA for their transcription. No GSII was found in the glnA ntrA mutant, and when a translational fusion of glnII to the Escherichia coli lacZ gene was introduced into WSU414, no beta-galactosidase was expressed. These results indicate that ntrA is required for glnII expression. The ntrA mutation did not prevent the expression of GSI. In free-living culture, the level of GSII and of the glnII-lacZ fusion protein was regulated by altering transcription in response to available nitrogen. No GSII protein was detected in alfalfa, pea, or soybean nodules when anti-GSII-specific antiserum was used. Images PMID:2570059

Genetic Code Optimization for Cotranslational Protein Folding: Codon Directional Asymmetry Correlates with Antiparallel Betasheets, tRNA Synthetase Classes.

PubMed

Seligmann, Hervé; Warthi, Ganesh

2017-01-01

A new codon property, codon directional asymmetry in nucleotide content (CDA), reveals a biologically meaningful genetic code dimension: palindromic codons (first and last nucleotides identical, codon structure XZX) are symmetric (CDA = 0), codons with structures ZXX/XXZ are 5'/3' asymmetric (CDA = - 1/1; CDA = - 0.5/0.5 if Z and X are both purines or both pyrimidines, assigning negative/positive (-/+) signs is an arbitrary convention). Negative/positive CDAs associate with (a) Fujimoto's tetrahedral codon stereo-table; (b) tRNA synthetase class I/II (aminoacylate the 2'/3' hydroxyl group of the tRNA's last ribose, respectively); and (c) high/low antiparallel (not parallel) betasheet conformation parameters. Preliminary results suggest CDA-whole organism associations (body temperature, developmental stability, lifespan). Presumably, CDA impacts spatial kinetics of codon-anticodon interactions, affecting cotranslational protein folding. Some synonymous codons have opposite CDA sign (alanine, leucine, serine, and valine), putatively explaining how synonymous mutations sometimes affect protein function. Correlations between CDA and tRNA synthetase classes are weaker than between CDA and antiparallel betasheet conformation parameters. This effect is stronger for mitochondrial genetic codes, and potentially drives mitochondrial codon-amino acid reassignments. CDA reveals information ruling nucleotide-protein relations embedded in reversed (not reverse-complement) sequences (5'-ZXX-3'/5'-XXZ-3').

New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.)

PubMed Central

Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; Ma, Xinming; Theg, Steven M.

2015-01-01

Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development. PMID:26307137

New isoforms and assembly of glutamine synthetase in the leaf of wheat ( Triticum aestivum L.)

DOE PAGES

Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; ...

2015-08-24

Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat ( Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSIImore » and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Lastly, our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.« less

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

PubMed

Matikainen, S; Ronni, T; Lehtonen, A; Sareneva, T; Melén, K; Nordling, S; Levy, D E; Julkunen, I

1997-06-01

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Mutations in MARS identified in a specific type of pulmonary alveolar proteinosis alter methionyl-tRNA synthetase activity.

PubMed

Comisso, Martine; Hadchouel, Alice; de Blic, Jacques; Mirande, Marc

2018-05-18

Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme. The pathophysiological mechanisms involved in MARS-related PAP are currently unknown. In this work, we analyzed the effect of the PAP-related mutations in MARS on the thermal stability and on the catalytic parameters of the MetRS mutants, relative to wild-type. The effect of these mutations on the structural integrity of the enzyme as a member of the cytosolic multisynthetase complex was also investigated. Our results establish that the PAP-related substitutions in MetRS impact the tRNA Met -aminoacylation reaction especially at the level of methionine recognition, and suggest a direct link between the loss of activity of the enzyme and the pathological disorders in PAP. © 2018 Federation of European Biochemical Societies.

Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast

NASA Technical Reports Server (NTRS)

Satyanarayana, T.; Mandel, A. D.; Klein, H. P.

1974-01-01

Evidence is presented that clearly establishes the presence of two acetyl-CoA synthetases in Saccharomyces cerevisiae, one elaborated under 'aerobic' conditions, the other under 'nonaerobic' conditions. The antibody produced by each enzyme is immunologically specific.

CDP-Diacylglycerol Synthetase Coordinates Cell Growth and Fat Storage through Phosphatidylinositol Metabolism and the Insulin Pathway

PubMed Central

Liu, Yuan; Wang, Wei; Shui, Guanghou; Huang, Xun

2014-01-01

During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi. PMID:24603715

Sequestration of auxin by the indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening.

PubMed

Böttcher, Christine; Keyzers, Robert A; Boss, Paul K; Davies, Christopher

2010-08-01

In fleshy fruit, levels of indole-3-acetic acid (IAA), the most abundant auxin, decline towards the onset of ripening. The application of auxins to immature fruit can delay the ripening processes. However, the mechanisms by which the decrease in endogenous IAA concentrations and the maintenance of low auxin levels in maturing fruit are achieved remain elusive. The transcript of a GH3 gene (GH3-1), encoding for an IAA-amido synthetase which conjugates IAA to amino acids, was detected in grape berries (Vitis vinifera L.). GH3-1 expression increased at the onset of ripening (veraison), suggesting that it might be involved in the establishment and maintenance of low IAA concentrations in ripening berries. Furthermore, this grapevine GH3 gene, responded positively to the combined application of abscisic acid and sucrose and to ethylene, linking it to the control of ripening processes. Levels of IAA-aspartic acid (IAA-Asp), an in vitro product of recombinant GH3-1, rose after veraison and remained high during the following weeks of the ripening phase when levels of free IAA were low. A similar pattern of changes in free IAA and IAA-Asp levels was detected in developing tomatoes (Solanum lycopersicum Mill.), where low concentrations of IAA and an increase in IAA-Asp concentrations coincided with the onset of ripening in this climacteric fruit. Since IAA-Asp might be involved in IAA degradation, the GH3 catalysed formation of this conjugate at, and after, the onset of ripening could represent a common IAA inactivation mechanism in climacteric and non-climacteric fruit which enables ripening.

The prokaryotic FAD synthetase family: a potential drug target.

PubMed

Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

2013-01-01

Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

Glutamic Acid as a Precursor to N-Terminal Pyroglutamic Acid in Mouse Plasmacytoma Protein

PubMed Central

Twardzik, Daniel R.; Peterkofsky, Alan

1972-01-01

Cell suspensions derived from a mouse plasmacytoma (RPC-20) that secretes an immunoglobulin light chain containing N-terminal pyroglutamic acid can synthesize protein in vitro. Chromatographic examination of an enzymatic digest of protein labeled with glutamic acid shows only labeled glutamic acid and pyroglutamic acid; hydrolysis of protein from cells labeled with glutamine, however, yields substantial amounts of glutamic acid in addition to glutamine and pyroglutamic acid. The absence of glutamine synthetase and presence of glutaminase in plasmacytoma homogenates is consistent with these findings. These data indicate that N-terminal pyroglutamic acid can be derived from glutamic acid without prior conversion of glutamic acid to glutamine. Since free or bound forms of glutamine cyclize nonezymatically to pyroglutamate with ease, while glutamic acid does not, the data suggest that N-terminal pyroglutamic acid formation from glutamic acid is enzymatic rather than spontaneous. Images PMID:4400295

Response to nitrate/ammonium nutrition of tomato (Solanum lycopersicum L.) plants overexpressing a prokaryotic NH4(+)-dependent asparagine synthetase.

PubMed

Martínez-Andújar, Cristina; Ghanem, Michel Edmond; Albacete, Alfonso; Pérez-Alfocea, Francisco

2013-05-01

Nitrogen availability is an important limiting factor for plant growth. Although NH4(+) assimilation is energetically more favorable than NO3(-), it is usually toxic for plants. In order to study if an improved ammonium assimilatory metabolism could increase the plant tolerance to ammonium nutrition, tomato (Solanum lycopersicum L. cv P-73) plants were transformed with an NH4(+)-dependent asparagine synthetase (AS-A) gene from Escherichia coli (asnA) under the control of a PCpea promoter (pea isolated constitutive promotor). Homozygous (Hom), azygous (Az) asnA and wild type (WT) plants were grown hydroponically for 6 weeks with normal Hoagland nutrition (NO3(-)/NH4(+)=6/0.5) and high ammonium nutrition (NO3(-)/NH4(+)=3.5/3). Under Hoagland's conditions, Hom plants produced 40-50% less biomass than WT and Az plants. However, under NO3(-)/NH4(+)=3.5/3 the biomass of Hom was not affected while it was reduced by 40-70% in WT and Az plants compared to Hoagland, respectively. The Hom plants accumulated 1.5-4 times more asparagine, glycine, serine and soluble proteins and registered higher glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in the light-adapted leaves than the other genotypes, but had similar NH4(+) and NO3(-) levels in all conditions. In the dark-adapted leaves, a protein catabolism occurred in the Hom plants with a concomitant 25-40% increase in organic acid concentration, while asparagine accumulation registered the highest values. The aforementioned processes might be responsible for a positive energetic balance as regards the futile cycle of the transgenic protein synthesis and catabolism. This explains growth penalty under standard nutrition and growth stability under NO3(-)/NH4(+)=3.5/3, respectively. Copyright © 2013 Elsevier GmbH. All rights reserved.

Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

PubMed

Kurylo-Borowska, Z

1975-07-14

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.

Succinyl-CoA synthetase (SUCLA2) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion.

PubMed

Huang, Xiaoping; Bedoyan, Jirair K; Demirbas, Didem; Harris, David J; Miron, Alexander; Edelheit, Simone; Grahame, George; DeBrosse, Suzanne D; Wong, Lee-Jun; Hoppel, Charles L; Kerr, Douglas S; Anselm, Irina; Berry, Gerard T

2017-03-01

Mutations in SUCLA2 result in succinyl-CoA ligase (ATP-forming) or succinyl-CoA synthetase (ADP-forming) (A-SCS) deficiency, a mitochondrial tricarboxylic acid cycle disorder. The phenotype associated with this gene defect is largely encephalomyopathy. We describe two siblings compound heterozygous for SUCLA2 mutations, c.985A>G (p.M329V) and c.920C>T (p.A307V), with parents confirmed as carriers of each mutation. We developed a new LC-MS/MS based enzyme assay to demonstrate the decreased SCS activity in the siblings with this unique genotype. Both siblings shared bilateral progressive hearing loss, encephalopathy, global developmental delay, generalized myopathy, and dystonia with choreoathetosis. Prior to diagnosis and because of lactic acidosis and low activity of muscle pyruvate dehydrogenase complex (PDC), sibling 1 (S1) was placed on dichloroacetate, while sibling 2 (S2) was on a ketogenic diet. S1 developed severe cyclic vomiting refractory to therapy, while S2 developed Leigh syndrome, severe GI dysmotility, intermittent anemia, hypogammaglobulinemia and eventually succumbed to his disorder. The mitochondrial DNA contents in skeletal muscle (SM) were normal in both siblings. Pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and several mitochondrial electron transport chain (ETC) activities were low or at the low end of the reference range in frozen SM from S1 and/or S2. In contrast, activities of PDC, other mitochondrial enzymes of pyruvate metabolism, ETC and, integrated oxidative phosphorylation, in skin fibroblasts were not significantly impaired. Although we show that propionyl-CoA inhibits PDC, it does not appear to account for decreased PDC activity in SM. A better understanding of the mechanisms of phenotypic variability and the etiology for tissue-specific secondary deficiencies of mitochondrial enzymes of oxidative metabolism, and independently mitochondrial DNA depletion (common in other cases of A-SCS deficiency), is needed

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

PubMed Central

Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

2015-01-01

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

Orthotopic Liver Transplantation for Urea Cycle Enzyme Deficiency

PubMed Central

Todo, Satoru; Starzl, Thomas E.; Tzakis, Andreas; Benkov, Keith J.; Kalousek, Frantisek; Saheki, Takeyori; Tanikawa, Kyuichi; Fenton, Wayne A.

2010-01-01

Hyperammonemia, abnormalities in plasma amino acids and abnormalities of standard liver functions were corrected by orthotopic liver transplantation in a 14-day-old boy with carbamyl phosphate synthetase-I deficiency and in a 35-yr-old man with argininosuccinic acid synthetase deficiency. The first patient had high plasma glutamine levels and no measureable citrulline, whereas citrulline values were markedly increased in Patient 2. Enzyme analysis of the original livers showed undetectable activity of carbamyl phosphate synthetase-I in Patient 1 and arginosuccinic acid synthetase in Patient 2. Both patients were comatose before surgery. Intellectual recovery of patient 1 has been slightly retarded because of a brain abscess caused by Aspergillus infection after surgery. Both patients are well at 34 and 40 mo, respectively, after surgery. Our experience has shown that orthotopic liver transplantation corrects the life-threatening metabolic abnormalities caused by deficiencies in the urea cycle enzymes carbamyl phosphate synthetase-I and arginosuccinic acid synthetase. Seven other patients–six with ornithine transcarbamylase deficiency and another with carbamyl phosphate synthetase-I deficiency–are known to have been treated elsewhere with liver transplantation 1½ yr or longer ago. Four of these seven recipients also are well, with follow-ups of 1½ to 5 yr. Thus liver transplantation corrects the metabolic abnormalities of three of the six urea cycle enzyme deficiencies, and presumably would correct all. PMID:1544622

The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.

PubMed

Herman, Jonathan D; Pepper, Lauren R; Cortese, Joseph F; Estiu, Guillermina; Galinsky, Kevin; Zuzarte-Luis, Vanessa; Derbyshire, Emily R; Ribacke, Ulf; Lukens, Amanda K; Santos, Sofia A; Patel, Vishal; Clish, Clary B; Sullivan, William J; Zhou, Huihao; Bopp, Selina E; Schimmel, Paul; Lindquist, Susan; Clardy, Jon; Mota, Maria M; Keller, Tracy L; Whitman, Malcolm; Wiest, Olaf; Wirth, Dyann F; Mazitschek, Ralph

2015-05-20

The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials. Copyright © 2015, American Association for the Advancement of Science.

Evolutionary divergence of chloroplast FAD synthetase proteins

PubMed Central

2010-01-01

Background Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity. PMID:20955574

Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

PubMed Central

Fang, Pengfei; Guo, Min

2015-01-01

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories. PMID:26670257

Statistical Evaluation of the Rodin–Ohno Hypothesis: Sense/Antisense Coding of Ancestral Class I and II Aminoacyl-tRNA Synthetases

PubMed Central

Chandrasekaran, Srinivas Niranj; Yardimci, Galip Gürkan; Erdogan, Ozgün; Roach, Jeffrey; Carter, Charles W.

2013-01-01

We tested the idea that ancestral class I and II aminoacyl-tRNA synthetases arose on opposite strands of the same gene. We assembled excerpted 94-residue Urgenes for class I tryptophanyl-tRNA synthetase (TrpRS) and class II Histidyl-tRNA synthetase (HisRS) from a diverse group of species, by identifying and catenating three blocks coding for secondary structures that position the most highly conserved, active-site residues. The codon middle-base pairing frequency was 0.35 ± 0.0002 in all-by-all sense/antisense alignments for 211 TrpRS and 207 HisRS sequences, compared with frequencies between 0.22 ± 0.0009 and 0.27 ± 0.0005 for eight different representations of the null hypothesis. Clustering algorithms demonstrate further that profiles of middle-base pairing in the synthetase antisense alignments are correlated along the sequences from one species-pair to another, whereas this is not the case for similar operations on sets representing the null hypothesis. Most probable reconstructed sequences for ancestral nodes of maximum likelihood trees show that middle-base pairing frequency increases to approximately 0.42 ± 0.002 as bacterial trees approach their roots; ancestral nodes from trees including archaeal sequences show a less pronounced increase. Thus, contemporary and reconstructed sequences all validate important bioinformatic predictions based on descent from opposite strands of the same ancestral gene. They further provide novel evidence for the hypothesis that bacteria lie closer than archaea to the origin of translation. Moreover, the inverse polarity of genetic coding, together with a priori α-helix propensities suggest that in-frame coding on opposite strands leads to similar secondary structures with opposite polarity, as observed in TrpRS and HisRS crystal structures. PMID:23576570

Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

PubMed

Hussain, Tahir; Yogavel, Manickam; Sharma, Amit

2015-04-01

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Inhibition of poly (ADP-ribose) Synthetase Attenuates Neutrophil Recruitment and Exerts Antiinflammatory Effects

PubMed Central

Szabó, Csaba; Lim, Lina H.K.; Cuzzocrea, Salvatore; Getting, Stephen J.; Zingarelli, Basilia; Flower, Roderick J.; Salzman, Andrew L.; Perretti, Mauro

1997-01-01

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10–20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS−/− mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity. PMID:9314553

Dietary Carcinogens and Breast Cancer.

DTIC Science & Technology

1997-07-01

11) proposed that L-proyl-tRNA synthetase could esterify N-hydroxy heterocyclic amines and that ATP was required to add the amino acid to the 16...need to be depleted of endogenous amino acids . To test this notion, tRNA synthetase substrates were removed by dialysis in one experiment, and activation...carcinogens in pyrolysates of amino acids and proteins and cooked foods: heterocyclic aromatic amines. In: Y.T. Woo (ed.) Chemical Induction of Cancer

Gene encoding plant asparagine synthetase

DOEpatents

Coruzzi, Gloria M.; Tsai, Fong-Ying

1993-10-26

The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

Characterization of long-chain acyl-CoA synthetases which stimulate secretion of fatty acids in green algae Chlamydomonas reinhardtii.

PubMed

Jia, Bin; Song, Yanzi; Wu, Min; Lin, Baicheng; Xiao, Kang; Hu, Zhangli; Huang, Ying

2016-01-01

Microalgae biofuel has become the most promising renewable energy over the past few years. But limitations still exist because of its high cost. Although, efforts have been made in enhancement of lipid productivity, the major cost problem in harvesting and oil extraction is still intractable. Thus, the idea of fatty acids (FAs) secretion which can massively facilitate algae harvesting and oil extraction was investigated here. The cDNAs of two long-chain acyl-CoA synthetases (LACSs) genes were cloned from Chlamydomonas reinhardtii and named as cracs1 and cracs2. They showed different substrate adaptation in the yeast complementation experiments. Cracs2 could utilize FAs C12:0, C14:0, C16:0, C18:0, C16:1 and C18:1, while crac1 could only utilize substrate C14:0, C16:1 and C18:1. Knockdown of cracs1 and cracs2 in C. reinhardtii resulted in accumulation of intracellular lipids. The total intracellular lipids contents of transgenic algae q-15 (knockdown of cracs1) and p-13 (knockdown of cracs2) were 45 and 55 %, respectively higher than that of cc849. Furthermore, FAs secretion was discovered in both transgenic algae. Secreted FAs can reach 8.19 and 9.66 mg/10(9) cells in q-15 and p-13, respectively. These results demonstrated the possibility of FAs secretion by microalgae and may give a new strategy of low-cost oil extraction. According to our findings, we proposed that FAs secretion may also be achieved in other species besides Chlamydomonas reinhardtii by knocking-down cracs genes, which may promote the future industrial application of microalgae biofuels.

Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin.

PubMed

van der Sluis, Rencia; Erasmus, Elardus

2016-10-01

Activation of fatty acids by the acyl-CoA synthetases (ACSs) is the vital first step in fatty acid metabolism. The enzymatic and physiological characterization of the human xenobiotic/medium chain fatty acid: CoA ligases (ACSMs) has been severely neglected even though xenobiotics, such as benzoate and salicylate, are detoxified through this pathway. This review will focus on the nomenclature and substrate specificity of the human ACSM ligases; the biochemical and enzymatic characterization of ACSM1 and ACSM2B; the high sequence homology of the ACSM2 genes (ACSM2A and ACSM2B) as well as what is currently known regarding disease association studies. Several discrepancies exist in the current literature that should be taken note of. For example, the single nucleotide polymorphisms (SNPs) reported to be associated with aspirin metabolism and multiple risk factors of metabolic syndrome are incorrect. Kinetic data on the substrate specificity of the human ACSM ligases are non-existent and currently no data exist on the influence of SNPs on the enzyme activity of these ligases. One of the biggest obstacles currently in the field is that glycine conjugation is continuously studied as a one-step process, which means that key regulatory factors of the two individual steps remain unknown.

Engineering Sialic Acid Synthesis Ability in Insect Cells.

PubMed

Viswanathan, Karthik; Narang, Someet; Betenbaugh, Michael J

2015-01-01

Insect cells lack the ability to synthesize the sialic acid donor molecule CMP-sialic acid or its precursor, sialic acid. In this chapter, we describe a method to engineer CMP-sialic acid synthesis capability into Spodoptera frugiperda (Sf9) cells, a prototypical insect cell line, by recombinant expression of sialic acid synthesis pathway genes using baculovirus technology. Co-expression of a sialuria mutant UDP-GlcNAc-2-epimerase/ManNAc kinase (EKR263L), wild-type sialic acid 9-phosphate synthase (SAS), and wild-type CMP-sialic acid synthetase (CSAS) in the presence of GlcNAc leads to synthesis of CMP-sialic acids synthesis to support sialylation of N-glycans on glycoproteins.

Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

PubMed

McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

2017-07-07

NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Clinical findings and effect of sodium hydrogen carbonate in patients with glutathione synthetase deficiency.

PubMed

Gündüz, Mehmet; Ünal, Özlem; Kavurt, Sumru; Türk, Emrecan; Mungan, Neslihan Önenli

2016-04-01

Glutathione synthetase (GS) deficiency is a rare inborn error of glutathione (GSH) metabolism manifested by severe metabolic acidosis, hemolytic anemia, neurological problems and massive excretion of pyroglutamic acid (5-oxoproline) in the urine. The disorder has mild, moderate, and severe clinical variants. We aimed to report clinical and laboratory findings of four patients, effect of sodium hydrogen carbonate treatment and long-term follow up of three patients. Urine organic acid analysis was performed with gas chromatography-mass spectrometry. Molecular genetic analysis was performed in three patients, mutation was found in two of them. Enzyme analysis was performed in one patient. Clinical and laboratory findings of four patients were evaluated. One patient died at 4 months old, one patient's growth and development are normal, two patients have developed intellectual disability and seizures in the long term follow up period. Three patients benefited from sodium hydrogen carbonate treatment. The clinical picture varies from patient to patient, so it is difficult to predict the prognosis and the effectiveness of treatment protocols. We reported long term follow up of four patients and demonstrated that sodium hydrogen carbonate is effective for treatment of chronic metabolic acidosis in GS deficieny.

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation.

PubMed

Nureki, Osamu; O'Donoghue, Patrick; Watanabe, Nobuhisa; Ohmori, Atsuhiko; Oshikane, Hiroyuki; Araiso, Yuhei; Sheppard, Kelly; Söll, Dieter; Ishitani, Ryuichiro

2010-11-01

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA(Gln). The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA(Glu) and Glu-tRNA(Gln). The Glu-tRNA(Gln) is then converted to Gln-tRNA(Gln) by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA(Glu) and tRNA(Gln) with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA(Glu)/tRNA(Gln) discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA(Gln) complex reveals the structural determinants responsible for specific tRNA(Gln) recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

PubMed

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of human cytosolic phenylalanyl-tRNA synthetase: evidence for kingdom-specific design of the active sites and tRNA binding patterns.

PubMed

Finarov, Igal; Moor, Nina; Kessler, Naama; Klipcan, Liron; Safro, Mark G

2010-03-10

The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs. We report herein the crystal structure of hcPheRS in complex with phenylalanine at 3.3 A resolution. A novel structural module has been revealed at the N terminus of the alpha subunit. It stretches out into the solvent of approximately 80 A and is made up of three structural domains (DBDs) possessing DNA-binding fold. The dramatic reduction of aminoacylation activity for truncated N terminus variants coupled with structural data and tRNA-docking model testify that DBDs play crucial role in hcPheRS activity.

MD Simulations of tRNA and Aminoacyl-tRNA Synthetases: Dynamics, Folding, Binding, and Allostery

PubMed Central

Li, Rongzhong; Macnamara, Lindsay M.; Leuchter, Jessica D.; Alexander, Rebecca W.; Cho, Samuel S.

2015-01-01

While tRNA and aminoacyl-tRNA synthetases are classes of biomolecules that have been extensively studied for decades, the finer details of how they carry out their fundamental biological functions in protein synthesis remain a challenge. Recent molecular dynamics (MD) simulations are verifying experimental observations and providing new insight that cannot be addressed from experiments alone. Throughout the review, we briefly discuss important historical events to provide a context for how far the field has progressed over the past few decades. We then review the background of tRNA molecules, aminoacyl-tRNA synthetases, and current state of the art MD simulation techniques for those who may be unfamiliar with any of those fields. Recent MD simulations of tRNA dynamics and folding and of aminoacyl-tRNA synthetase dynamics and mechanistic characterizations are discussed. We highlight the recent successes and discuss how important questions can be addressed using current MD simulations techniques. We also outline several natural next steps for computational studies of AARS:tRNA complexes. PMID:26184179

Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade.

PubMed Central

Varón-Castellanos, R; Havsteen, B H; García-Moreno, M; Valero-Ruiz, E; Molina-Alarcón, M; García-Cánovas, F

1993-01-01

A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived. PMID:8104399

Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity1[OPEN

PubMed Central

Pedersen, Carsten

2016-01-01

Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of the high-affinity low-capacity isoform Gln-1;1 was reduced. The expression of Gln-1;3 did not respond to ammonium treatment while Gln-1;4 and Gln-1;5 isogenes in all cases were expressed at a very low level. Gln-2 was highly expressed in shoots but only at a very low level in roots. To investigate the specific functions of the two isogenes Gln-1;1 and Gln-1;2 in shoots for ammonium detoxification, single and double knock-out mutants were grown under standard N supply or with high ammonium provision. Phenotypes of the single mutant gln1;1 were similar to the wild type, while growth of the gln1;2 single mutant and the gln1;1:gln1;2 double mutant was significantly impaired irrespective of N regime. GS1 activity was significantly reduced in both gln1;2 and gln1;1:gln1;2. Along with this, the ammonium content increased while that of Gln decreased, showing that Gln-1;2 was essential for ammonium assimilation and amino acid synthesis. We conclude that Gln-1;2 is the main isozyme contributing to shoot GS1 activity in vegetative growth stages and can be up-regulated to relieve ammonium toxicity. This reveals, to our knowledge, a novel shoot function of Gln-1;2 in Arabidopsis shoots. PMID:27231101

Evidence for symbiont-induced alteration of a host's gene expression: irreversible loss of SAM synthetase from Amoeba proteus.

PubMed

Choi, J Y; Lee, T W; Jeon, K W; Ahn, T I

1997-01-01

Symbiont-bearing xD amoebae no longer produce a 45-kDa cytoplasmic protein that functions as S-adenosylmethionine synthetase in symbiont-free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X-bacteria supply the needed enzyme. A monoclonal antibody against the 45-kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S-adenosylmethionine synthetase activity. These results show how symbiotic X-bacteria may become essential cellular components of amoeba by supplementing a genetic defect for an amoeba's house-keeping gene that is brought about by an action of X-bacteria themselves. This is the first reported example in which symbionts alter the host's gene expression to block the production of an essential protein.

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.

PubMed

Fan, Chenguang; Xiong, Hai; Reynolds, Noah M; Söll, Dieter

2015-12-15

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA(Pyl) to create tRNA(Pyl-opt) with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N(ϵ)-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA(Pyl-opt) facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA(Pyl-opt) with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA(Pyl-opt) had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA(Pyl-opt) should be an excellent replacement of wild-type tRNA(Pyl) for future ncAA incorporation by PylRS enzymes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Twin Attributes of Tyrosyl-tRNA Synthetase of Leishmania donovani: A HOUSEKEEPING PROTEIN TRANSLATION ENZYME AND A MIMIC OF HOST CHEMOKINE.

PubMed

Anand, Sneha; Madhubala, Rentala

2016-08-19

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes essential for protein synthesis. Apart from their parent aminoacylation activity, several aaRSs perform non-canonical functions in diverse biological processes. The present study explores the twin attributes of Leishmania tyrosyl-tRNA synthetase (LdTyrRS) namely, aminoacylation, and as a mimic of host CXC chemokine. Leishmania donovani is a protozoan parasite. Its genome encodes a single copy of tyrosyl-tRNA synthetase. We first tested the canonical aminoacylation role of LdTyrRS. The recombinant protein was expressed, and its kinetic parameters were determined by aminoacylation assay. To study the physiological role of LdTyrRS in Leishmania, gene deletion mutations were attempted via targeted gene replacement. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. LdTyrRS appears to be an essential gene as the chromosomal null mutants did not survive. Our data also highlights the non-canonical function of L. donovani tyrosyl-tRNA synthetase. We show that LdTyrRS protein is present in the cytoplasm and exits from the parasite cytoplasm into the extracellular medium. The released LdTyrRS functions as a neutrophil chemoattractant. We further show that LdTyrRS specifically binds to host macrophages with its ELR (Glu-Leu-Arg) peptide motif. The ELR-CXCR2 receptor interaction mediates this binding. This interaction triggers enhanced secretion of the proinflammatory cytokines TNF-α and IL-6 by host macrophages. Our data indicates a possible immunomodulating role of LdTyrRS in Leishmania infection. This study provides a platform to explore LdTyrRS as a potential target for drug development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molybdenum Involvement in Aerobic Degradation of 2-Furoic Acid by Pseudomonas putida Fu1

PubMed Central

Koenig, Kerstin; Andreesen, Jan Remmer

1989-01-01

An organism identified as Pseudomonas putida was isolated from an enrichment culture with 2-furoic acid as its sole source of carbon and energy. The organism contained a 2-furoyl-coenzyme A (CoA) synthetase to form 2-furoyl-CoA and a 2-furoyl-CoA dehydrogenase to form 5-hydroxy-2-furoyl-CoA as the first two enzymes involved in the degradation. Tungstate, the specific antagonist of molybdate, decreased growth rate and consumption of 2-furoic acid but had no influence on growth with succinate. Correspondingly, the 2-furoyl-CoA dehydrogenase activity decreased when the organism was grown on 2-furoic acid in the presence of increasing amounts of tungstate. The addition of molybdate reversed the negative effect on 2-furoyl-CoA dehydrogenase activity, which points to the involvement of a molybdoenzyme in this reaction. Both enzymes studied were inducible. No plasmid was detected in this organism. PMID:16347977

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus.

PubMed

Sun, Wei-Wen; Guo, Chun-Jun; Wang, Clay C C

2016-04-01

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity

PubMed Central

Beyer, Dieter; Kroll, Hein-Peter; Endermann, Rainer; Schiffer, Guido; Siegel, Stephan; Bauser, Marcus; Pohlmann, Jens; Brands, Michael; Ziegelbauer, Karl; Haebich, Dieter; Eymann, Christine; Brötz-Oesterhelt, Heike

2004-01-01

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNAPhe), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 μg/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNAPhe charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA+ parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy. PMID:14742205

Intracellular synthesis of glutamic acid in Bacillus methylotrophicus SK19.001, a glutamate-independent poly(γ-glutamic acid)-producing strain.

PubMed

Peng, Yingyun; Zhang, Tao; Mu, Wanmeng; Miao, Ming; Jiang, Bo

2016-01-15

Bacillus methylotrophicus SK19.001 is a glutamate-independent strain that produces poly(γ-glutamic acid) (γ-PGA), a polymer of D- and L-glutamic acids that possesses applications in food, the environment, agriculture, etc. This study was undertaken to explore the synthetic pathway of intracellular L- and D-glutamic acid in SK19.001 by investigating the effects of tricarboxylic acid cycle intermediates and different amino acids as metabolic precursors on the production of γ-PGA and analyzing the activities of the enzymes involved in the synthesis of L- and D-glutamate. Tricarboxylic acid cycle intermediates and amino acids could participate in the synthesis of γ-PGA via independent pathways in SK19.001. L-Aspartate aminotransferase, L-glutaminase and L-glutamate synthase were the enzymatic sources of L-glutamate. Glutamate racemase was responsible for the formation of D-glutamate for the synthesis of γ-PGA, and the synthetase had stereoselectivity for glutamate substrate. The enzymatic sources of L-glutamate were investigated for the first time in the glutamate-independent γ-PGA-producing strain, and multiple enzymatic sources of L-glutamate were verified in SK19.001, which will benefit efforts to improve production of γ-PGA with metabolic engineering strategies. © 2015 Society of Chemical Industry.

3-Methylglutaconic aciduria, a frequent but underrecognized finding in carbamoyl phosphate synthetase I deficiency.

PubMed

Rokicki, Dariusz; Pajdowska, Magdalena; Trubicka, Joanna; Thong, Meow-Keong; Ciara, Elżbieta; Piekutowska-Abramczuk, Dorota; Pronicki, Maciej; Sikora, Roman; Haidar, Rijad; Ołtarzewski, Mariusz; Jabłońska, Ewa; Muthukumarasamy, Premala; Sthaneswar, Pavai; Gan, Chin-Seng; Krajewska-Walasek, Małgorzata; Carrozzo, Rosalba; Verrigni, Daniela; Semeraro, Michela; Rizzo, Cristiano; Taurisano, Roberta; Alhaddad, Bader; Kovacs-Nagy, Reka; Haack, Tobias B; Dionisi-Vici, Carlo; Pronicka, Ewa; Wortmann, Saskia B

2017-08-01

The urea cycle disorder carbamoyl phosphate synthetase I deficiency is an important differential diagnosis in the encephalopathic neonate. This intoxication type inborn error of metabolism often leads to neonatal death or severe and irreversible damage of the central nervous system, even despite appropriate treatment. Timely diagnosis is crucial, but can be difficult on routine metabolite level. Here, we report ten neonates from eight families (finally) diagnosed with CPS1 deficiency at three tertiary metabolic centres. In seven of them the laboratory findings were dominated by significantly elevated urinary 3-methylglutaconic acid levels which complicated the diagnostic process. Our findings are both important for the differential diagnosis of patients with urea cycle disorders and also broaden the differential diagnosis of hyperammonemia associated with 3-methylglutaconic aciduria, which was earlier only reported in TMEM70 and SERAC1 defect. Copyright © 2017 Elsevier B.V. All rights reserved.

Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells.

PubMed

Jastreboff, M M; Kedzierska, B; Rode, W

1983-07-15

Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.

Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

PubMed Central

Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

2015-01-01

Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

Inhibition effect of isopropanol on acetyl-CoA synthetase expression level of acetoclastic methanogen, Methanosaeta concilii.

PubMed

Ince, Bahar; Koksel, Gozde; Cetecioglu, Zeynep; Oz, Nilgun Ayman; Coban, Halil; Ince, Orhan

2011-11-10

Isopropanol is a widely found solvent in industrial wastewaters, which have commonly been treated using anaerobic systems. In this study, inhibitory effect of isopropanol on the key microbial group in anaerobic bioreactors, acetoclastic methanogens, was investigated. Anaerobic sludges in serum bottles were repeatedly fed with acetate and isopropanol; and quantitative real-time PCR was used for determining effect of isopropanol on the expression level of a key enzyme in acetoclastic methane production, acetyl-CoA synthetase of Methanosaeta concilii. Active Methanosaeta spp. cells were also quantified using Fluorescent in situ hybridization (FISH). Transcript abundance of acetyl-CoA synthetase was 1.23±0.62×10(6) mRNAs/mL in the uninhibited reactors with 222 mL cumulative methane production. First exposure to isopropanol resulted in 71.2%, 84.7%, 89.2% and 94.6% decrease in mRNA level and 35.0%, 65.0%, 91.5% and 100.0% reduction in methane production for isopropanol concentrations of 0.1 M, 0.5 M, 1.0 M and 2.0 M, respectively. Repeated exposures resulted in higher inhibitions; and at the end of test, fluorescent intensities of active Methanosaeta cells were significantly decreased due to isopropanol. The overall results indicated that isopropanol has an inhibitory effect on acetoclastic methanogenesis; and the inhibition can be detected by monitoring level of acetyl-CoA transcripts and rRNA level. Copyright © 2011 Elsevier B.V. All rights reserved.

Proximal tubule glutamine synthetase expression is necessary for the normal response to dietary protein restriction.

PubMed

Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Verlander, Jill W; Weiner, I David

2017-07-01

Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion changes in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of [Formula: see text] and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The purpose of the current study was to determine the role of proximal tubule GS in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low-protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule GS expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.

Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

PubMed

McCormack, M; Brecher, P

1987-06-15

Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes.

tRNAGlu increases the affinity of glutamyl-tRNA synthetase for its inhibitor glutamyl-sulfamoyl-adenosine, an analogue of the aminoacylation reaction intermediate glutamyl-AMP: mechanistic and evolutionary implications.

PubMed

Blais, Sébastien P; Kornblatt, Jack A; Barbeau, Xavier; Bonnaure, Guillaume; Lagüe, Patrick; Chênevert, Robert; Lapointe, Jacques

2015-01-01

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of -TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant Kd is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3'-OH oxygen of the 3'-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.

tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine, an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

PubMed Central

Blais, Sébastien P.; Kornblatt, Jack A.; Barbeau, Xavier; Bonnaure, Guillaume; Lagüe, Patrick; Chênevert, Robert; Lapointe, Jacques

2015-01-01

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of –TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant K d is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP. PMID:25860020

Isolation and characterization of the gene coding for Escherichia coli arginyl-tRNA synthetase.

PubMed Central

Eriani, G; Dirheimer, G; Gangloff, J

1989-01-01

The gene coding for Escherichia coli arginyl-tRNA synthetase (argS) was isolated as a fragment of 2.4 kb after analysis and subcloning of recombinant plasmids from the Clarke and Carbon library. The clone bearing the gene overproduces arginyl-tRNA synthetase by a factor 100. This means that the enzyme represents more than 20% of the cellular total protein content. Sequencing revealed that the fragment contains a unique open reading frame of 1734 bp flanked at its 5' and 3' ends respectively by 247 bp and 397 bp. The length of the corresponding protein (577 aa) is well consistent with earlier Mr determination (about 70 kd). Primer extension analysis of the ArgRS mRNA by reverse transcriptase, located its 5' end respectively at 8 and 30 nucleotides downstream of a TATA and a TTGAC like element (CTGAC) and 60 nucleotides upstream of the unusual translation initiation codon GUG; nuclease S1 analysis located the 3'-end at 48 bp downstream of the translation termination codon. argS has a codon usage pattern typical for highly expressed E. coli genes. With the exception of the presence of a HVGH sequence similar to the HIGH consensus element, ArgRS has no relevant sequence homologies with other aminoacyl-tRNA synthetases. Images PMID:2668891

A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in Aspergillus fumigatus.

PubMed

Reeves, Emer P; Reiber, Kathrin; Neville, Claire; Scheibner, Olaf; Kavanagh, Kevin; Doyle, Sean

2006-07-01

Aspergillus fumigatus is an important human fungal pathogen. The Aspergillus fumigatus genome contains 14 nonribosomal peptide synthetase genes, potentially responsible for generating metabolites that contribute to organismal virulence. Differential expression of the nonribosomal peptide synthetase gene, pes1, in four strains of Aspergillus fumigatus was observed. The pattern of pes1 expression differed from that of a putative siderophore synthetase gene, sidD, and so is unlikely to be involved in iron acquisition. The Pes1 protein (expected molecular mass 698 kDa) was partially purified and identified by immunoreactivity, peptide mass fingerprinting (36% sequence coverage) and MALDI LIFT-TOF/TOF MS (four internal peptides sequenced). A pes1 disruption mutant (delta pes1) of Aspergillus fumigatus strain 293.1 was generated and confirmed by Southern and western analysis, in addition to RT-PCR. The delta pes1 mutant also showed significantly reduced virulence in the Galleria mellonella model system (P < 0.001) and increased sensitivity to oxidative stress (P = 0.002) in culture and during neutrophil-mediated phagocytosis. In addition, the mutant exhibited altered conidial surface morphology and hydrophilicity, compared to Aspergillus fumigatus 293.1. It is concluded that pes1 contributes to improved fungal tolerance against oxidative stress, mediated by the conidial phenotype, during the infection process.

Arabidopsis plastidial folylpolyglutamate synthetase is required for seed reserve accumulation and seedling establishment in darkness.

PubMed

Meng, Hongyan; Jiang, Ling; Xu, Bosi; Guo, Wenzhu; Li, Jinglai; Zhu, Xiuqing; Qi, Xiaoquan; Duan, Lixin; Meng, Xianbin; Fan, Yunliu; Zhang, Chunyi

2014-01-01

Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB) was defective in seed reserves and skotomorphogenesis. Lower carbon (C) and higher nitrogen (N) content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3-. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3- conditions, and further enhanced under NO3- limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3- during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3- as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis.

Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

NASA Technical Reports Server (NTRS)

Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

1987-01-01

The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

[Influence of exogenous gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid contents in roots of melon seedling under hypoxia stress].

PubMed

Wang, Chun-Yan; Li, Jing-Rui; Xia, Qing-Ping; Wu, Xiao-Lei; Gao, Hong-Bo

2014-07-01

This paper investigated the influence of gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid content under hypoxia stress by accurately controlling the level of dissolved oxygen in hydroponics, using the roots of melon 'Xiyu 1' seedlings as the test material. The results showed that compared with the control, the growth of roots was inhibited seriously under hypoxia stress. Meanwhile, the hypoxia-treated roots had significantly higher activities of glutamate decarboxylase (GAD), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST) as well as the contents of GABA, pyruvic acid, alanine (Ala) and aspartic acid (Asp). But the contents of glutamic acid (Glu) and alpha-keto glutaric acid in roots under hypoxia stress was obviously lower than those of the control. Exogenous treatment with GABA alleviated the inhibition effect of hypoxia stress on root growth, which was accompanied by an increase in the contents of endogenous GABA, Glu, alpha-keto glutaric acid and Asp. Furthermore, under hypoxia stress, the activities of GAD, GDH, GOGAT, GS, ALT, AST as well as the contents of pyruvic acid and Ala significantly decreased in roots treated with GABA. However, adding GABA and viny-gamma-aminobutyric acid (VGB) reduced the alleviation effect of GABA on melon seedlings under hypoxia stress. The results suggested that absorption of GABA by roots could alleviate the injury of hypoxia stress to melon seedlings. This meant that GABA treatment allows the normal physiological metabolism under hypoxia by inhibiting the GAD activity through feedback and maintaining higher Glu content as well as the bal- ance of carbon and nitrogen.

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains

PubMed Central

Kassan, Adam; Herms, Albert; Fernández-Vidal, Andrea; Bosch, Marta; Schieber, Nicole L.; Reddy, Babu J.N.; Fajardo, Alba; Gelabert-Baldrich, Mariona; Tebar, Francesc; Enrich, Carlos; Gross, Steven P.

2013-01-01

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of “emerging” LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage. PMID:24368806

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

PubMed Central

Donini, Pierluigi

1970-01-01

Starvation for a required amino acid of normal or RCstrEscherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RCrelE. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RCstr phenotype but not in cells of RCrel phenotype. Inhibition of phage DNA synthesis in amino acid-starved RCstr host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought. PMID:4914067

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis.

PubMed

Donini, Stefano; Garavaglia, Silvia; Ferraris, Davide M; Miggiano, Riccardo; Mori, Shigetarou; Shibayama, Keigo; Rizzi, Menico

2017-01-01

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

Sex and depot differences in ex vivo adipose tissue fatty acid storage and glycerol-3-phosphate acyltransferase activity

PubMed Central

Morgan-Bathke, Maria; Chen, Liang; Oberschneider, Elisabeth; Harteneck, Debra

2015-01-01

Adipose tissue fatty acid storage varies according to sex, adipose tissue depot, and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We examined whether differences in adipose tissue glycerol-3-phosphate acyltransferase (GPAT) might play a role in these variations. We optimized an enzyme activity assay for total GPAT and GPAT1 activity in human adipose tissue and measured GPAT activity. Omental and subcutaneous adipose tissue was collected from obese and nonobese adults for measures of GPAT and GPAT1 activities, ex vivo palmitate storage, acyl-CoA synthetase (ACS) and diacylglycerol-acyltransferase (DGAT) activities, and CD36 protein. Total GPAT and GPAT1 activities decreased as a function of adipocyte size in both omental (r = −0.71, P = 0.003) and subcutaneous (r = −0.58, P = 0.04) fat. The relative contribution of GPAT1 to total GPAT activity increased as a function of adipocyte size, accounting for up to 60% of GPAT activity in those with the largest adipocytes. We found strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots (r values 0.58–0.91) and between these storage factors and palmitate storage rates into TAG (r values 0.55–0.90). We conclude that: 1) total GPAT activity decreases as a function of adipocyte size; 2) GPAT1 can account for over half of adipose GPAT activity in hypertrophic obesity; and 3) ACS, GPAT, and DGAT are coordinately regulated. PMID:25738782

The role of strong electrostatic interactions at the dimer interface of human glutathione synthetase.

PubMed

De Jesus, Margarita C; Ingle, Brandall L; Barakat, Khaldoon A; Shrestha, Bisesh; Slavens, Kerri D; Cundari, Thomas R; Anderson, Mary E

2014-10-01

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein-protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.

Archaea recruited D-Tyr-tRNATyr deacylase for editing in Thr-tRNA synthetase.

PubMed

Rigden, Daniel J

2004-12-01

Aminoacyl-tRNA synthetases (AARSs) are key players in the maintenance of the genetic code through correct pairing of amino acids with their cognate tRNA molecules. To this end, some AARSs, as well as seeking to recognize the correct amino acid during synthesis of aminoacyl-tRNA, enhance specificity through recognition of mischarged aminoacyl-tRNA molecules in a separate editing reaction. Recently, an editing domain, of uncertain provenance, idiosyncratic to some archaeal ThrRSs has been characterized. Here, sequence analyses and molecular modeling are reported that clearly show a relationship of the archaea-specific ThrRS editing domains with d-Tyr-tRNATyr deacylases (DTDs). The model enables the identification of the catalytic site and other substrate binding residues, as well as the proposal of a likely catalytic mechanism. Interestingly, typical DTD sequences, common in bacteria and eukaryotes, are entirely absent in archaea, consistent with an evolutionary scheme in which DTD was co-opted to serve as a ThrRS editing domain in archaea soon after their divergence from eukaryotes. A group of present-day archaebacteria contain a ThrRS obtained from a bacterium by horizontal gene transfer. In some of these cases a vestigial version of the original archaeal ThrRS, of potentially novel function, is maintained.

Archaea recruited d-Tyr-tRNATyr deacylase for editing in Thr–tRNA synthetase

PubMed Central

RIGDEN, DANIEL J.

2004-01-01

Aminoacyl–tRNA synthetases (AARSs) are key players in the maintenance of the genetic code through correct pairing of amino acids with their cognate tRNA molecules. To this end, some AARSs, as well as seeking to recognize the correct amino acid during synthesis of aminoacyl–tRNA, enhance specificity through recognition of mischarged aminoacyl–tRNA molecules in a separate editing reaction. Recently, an editing domain, of uncertain provenance, idiosyncratic to some archaeal ThrRSs has been characterized. Here, sequence analyses and molecular modeling are reported that clearly show a relationship of the archaea-specific ThrRS editing domains with d-Tyr-tRNATyr deacylases (DTDs). The model enables the identification of the catalytic site and other substrate binding residues, as well as the proposal of a likely catalytic mechanism. Interestingly, typical DTD sequences, common in bacteria and eukaryotes, are entirely absent in archaea, consistent with an evolutionary scheme in which DTD was co-opted to serve as a ThrRS editing domain in archaea soon after their divergence from eukaryotes. A group of present-day archaebacteria contain a ThrRS obtained from a bacterium by horizontal gene transfer. In some of these cases a vestigial version of the original archaeal ThrRS, of potentially novel function, is maintained. PMID:15525705

Synthesis and biological activity of amino acid conjugates of abscisic acid.

PubMed

Todoroki, Yasushi; Narita, Kenta; Muramatsu, Taku; Shimomura, Hajime; Ohnishi, Toshiyuki; Mizutani, Masaharu; Ueno, Kotomi; Hirai, Nobuhiro

2011-03-01

We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture*

PubMed Central

Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M.

2016-01-01

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. PMID:27597544

Use of Lantibiotic Synthetases for the Preparation of Bioactive Constrained Peptides

PubMed Central

Levengood, Matthew R.

2008-01-01

Stabilization of biologically active peptides is a major goal in peptide-based drug design. Cyclization is an often-used strategy to enhance resistance of peptides towards protease degradation and simultaneously improve their affinity for targets by restricting their conformational flexibility. Amongst the various cyclization strategies, the use of thioether crosslinks has been successful for various peptides including enkephalin. The synthesis of these thioethers can be arduous, especially for longer peptides. Described herein is an enzymatic strategy taking advantage of the lantibiotic synthetase LctM that dehydrates Ser and Thr residues to the corresponding dehydroalanine and dehydrobutyrine residues and catalyzes the Michael-type addition of Cys residues to form thioether crosslinks. The use of LctM to prepare thioether containing analogs of enkephalin, contryphan, and inhibitors of human tripeptidyl peptidase II and spider venom epimerase is demonstrated. PMID:18294843

Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway.

PubMed

Lee, Minji; Kim, Jong Hyun; Yoon, Ina; Lee, Chulho; Fallahi Sichani, Mohammad; Kang, Jong Soon; Kang, Jeonghyun; Guo, Min; Lee, Kang Young; Han, Gyoonhee; Kim, Sunghoon; Han, Jung Min

2018-06-05

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating "ON" switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an "OFF" switch by controlling GTP hydrolysis of RagB in the Rag GTPase-mTORC1 axis. The LRS-RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP-GDP cycle of the RagD-RagB pair, rather than the RagC-RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD-RagB pair can overcome the absence of the RagC-RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD-RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

Mutations of the aminoacyl-tRNA-synthetases SARS and WARS2 are implicated in the etiology of autosomal recessive intellectual disability.

PubMed

Musante, Luciana; Püttmann, Lucia; Kahrizi, Kimia; Garshasbi, Masoud; Hu, Hao; Stehr, Henning; Lipkowitz, Bettina; Otto, Sabine; Jensen, Lars R; Tzschach, Andreas; Jamali, Payman; Wienker, Thomas; Najmabadi, Hossein; Ropers, Hans Hilger; Kuss, Andreas W

2017-06-01

Intellectual disability (ID) is the hallmark of an extremely heterogeneous group of disorders that comprises a wide variety of syndromic and non-syndromic phenotypes. Here, we report on mutations in two aminoacyl-tRNA synthetases that are associated with ID in two unrelated Iranian families. In the first family, we identified a homozygous missense mutation (c.514G>A, p.Asp172Asn) in the cytoplasmic seryl-tRNA synthetase (SARS) gene. The mutation affects the enzymatic core domain of the protein and impairs its enzymatic activity, probably leading to reduced cytoplasmic tRNA Ser concentrations. The mutant protein was predicted to be unstable, which could be substantiated by investigating ectopic mutant SARS in transfected HEK293T cells. In the second family, we found a compound heterozygous genotype of the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, comprising a nonsense mutation (c.325delA, p.Ser109Alafs*15), which very likely entails nonsense-mediated mRNA decay and a missense mutation (c.37T>G, p.Trp13Gly). The latter affects the mitochondrial localization signal of WARS2, causing protein mislocalization. Including AIMP1, which we have recently implicated in the etiology of ID, three genes with a role in tRNA-aminoacylation are now associated with this condition. We therefore suggest that the functional integrity of tRNAs in general is an important factor in the development and maintenance of human cognitive functions. © 2017 Wiley Periodicals, Inc.

Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli.

PubMed

Bouhss, A; Dementin, S; van Heijenoort, J; Parquet, C; Blanot, D

1999-06-18

The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.

Spontaneous mutual ordering of nucleic acids and proteins.

PubMed

Wills, Peter R

2014-12-01

It is proposed that the prebiotic ordering of nucleic acid and peptide sequences was a cooperative process in which nearly random populations of both kinds of polymers went through a codependent series of self-organisation events that simultaneously refined not only the accuracy of genetic replication and coding but also the functional specificity of protein catalysts, especially nascent aminoacyl-tRNA synthetase "urzymes".

Revisiting the operational RNA code for amino acids: Ensemble attributes and their implications.

PubMed

Shaul, Shaul; Berel, Dror; Benjamini, Yoav; Graur, Dan

2010-01-01

It has been suggested that tRNA acceptor stems specify an operational RNA code for amino acids. In the last 20 years several attributes of the putative code have been elucidated for a small number of model organisms. To gain insight about the ensemble attributes of the code, we analyzed 4925 tRNA sequences from 102 bacterial and 21 archaeal species. Here, we used a classification and regression tree (CART) methodology, and we found that the degrees of degeneracy or specificity of the RNA codes in both Archaea and Bacteria differ from those of the genetic code. We found instances of taxon-specific alternative codes, i.e., identical acceptor stem determinants encrypting different amino acids in different species, as well as instances of ambiguity, i.e., identical acceptor stem determinants encrypting two or more amino acids in the same species. When partitioning the data by class of synthetase, the degree of code ambiguity was significantly reduced. In cryptographic terms, a plausible interpretation of this result is that the class distinction in synthetases is an essential part of the decryption rules for resolving the subset of RNA code ambiguities enciphered by identical acceptor stem determinants of tRNAs acylated by enzymes belonging to the two classes. In evolutionary terms, our findings lend support to the notion that in the pre-DNA world, interactions between tRNA acceptor stems and synthetases formed the basis for the distinction between the two classes; hence, ambiguities in the ancient RNA code were pivotal for the fixation of these enzymes in the genomes of ancestral prokaryotes.

Revisiting the operational RNA code for amino acids: Ensemble attributes and their implications

PubMed Central

Shaul, Shaul; Berel, Dror; Benjamini, Yoav; Graur, Dan

2010-01-01

It has been suggested that tRNA acceptor stems specify an operational RNA code for amino acids. In the last 20 years several attributes of the putative code have been elucidated for a small number of model organisms. To gain insight about the ensemble attributes of the code, we analyzed 4925 tRNA sequences from 102 bacterial and 21 archaeal species. Here, we used a classification and regression tree (CART) methodology, and we found that the degrees of degeneracy or specificity of the RNA codes in both Archaea and Bacteria differ from those of the genetic code. We found instances of taxon-specific alternative codes, i.e., identical acceptor stem determinants encrypting different amino acids in different species, as well as instances of ambiguity, i.e., identical acceptor stem determinants encrypting two or more amino acids in the same species. When partitioning the data by class of synthetase, the degree of code ambiguity was significantly reduced. In cryptographic terms, a plausible interpretation of this result is that the class distinction in synthetases is an essential part of the decryption rules for resolving the subset of RNA code ambiguities enciphered by identical acceptor stem determinants of tRNAs acylated by enzymes belonging to the two classes. In evolutionary terms, our findings lend support to the notion that in the pre-DNA world, interactions between tRNA acceptor stems and synthetases formed the basis for the distinction between the two classes; hence, ambiguities in the ancient RNA code were pivotal for the fixation of these enzymes in the genomes of ancestral prokaryotes. PMID:19952117

Fatty acid-binding protein 5 (FABP5) promotes lipolysis of lipid droplets, de novo fatty acid (FA) synthesis and activation of nuclear factor-kappa B (NF-κB) signaling in cancer cells.

PubMed

Senga, Shogo; Kobayashi, Narumi; Kawaguchi, Koichiro; Ando, Akira; Fujii, Hiroshi

2018-06-12

Fatty acid-binding proteins (FABPs) are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting them to the appropriate compartments in the cell. Epidermal fatty acid-binding protein (FABP5) is an intracellular lipid-binding protein that is abundantly expressed in adipocytes and macrophages. Previous studies have revealed that the FABP5 expression level is closely related to malignancy in various types of cancer. However, its precise functions in the metabolisms of cancer cells remain unclear. Here, we revealed that FABP5 knockdown significantly induced downregulation of the genes expression, such as hormone-sensitive lipase (HSL), monoacylglycerol lipase (MAGL), elongation of long-chain fatty acid member 6 (Elovl6), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in altered lipid metabolism, lipolysis, and de novo FA synthesis in highly aggressive prostate and breast cancer cells. Moreover, we demonstrated that FABP5 induced inflammation and cytokine production through the nuclear factor-kappa B signaling pathway activated by reactive oxygen species and protein kinase C in PC-3 and MDA-MB-231 cells. Thus, FABP5 might regulate lipid quality and/or quantity to promote aggressiveness such as cell growth, invasiveness, survival, and inflammation in prostate and breast cancer cells. In the present study, we have revealed for the first time that high expression of FABP5 plays a critical role in alterations of lipid metabolism, leading to cancer development and metastasis in highly aggressive prostate and breast cancer cells. Copyright © 2018. Published by Elsevier B.V.

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis

PubMed Central

Donini, Stefano; Garavaglia, Silvia; Ferraris, Davide M.; Miggiano, Riccardo; Mori, Shigetarou; Shibayama, Keigo

2017-01-01

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest. PMID:28419153

The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

PubMed

Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

2016-02-13

Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

PubMed

León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

2017-08-21

The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

PubMed Central

Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

1992-01-01

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

Arabidopsis Plastidial Folylpolyglutamate Synthetase Is Required for Seed Reserve Accumulation and Seedling Establishment in Darkness

PubMed Central

Meng, Hongyan; Jiang, Ling; Xu, Bosi; Guo, Wenzhu; Li, Jinglai; Zhu, Xiuqing; Qi, Xiaoquan; Duan, Lixin; Meng, Xianbin; Fan, Yunliu; Zhang, Chunyi

2014-01-01

Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB) was defective in seed reserves and skotomorphogenesis. Lower carbon (C) and higher nitrogen (N) content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3 −. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3 − conditions, and further enhanced under NO3 − limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3 − during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3 − as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis. PMID:25000295

ε-Poly-l-Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly-l-Lysine Synthetase

PubMed Central

Kito, Naoko; Kita, Akihiro; Imokawa, Yuuki; Yamanaka, Kazuya; Maruyama, Chitose; Katano, Hajime

2014-01-01

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL. PMID:24907331

ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.

PubMed

Hamano, Yoshimitsu; Kito, Naoko; Kita, Akihiro; Imokawa, Yuuki; Yamanaka, Kazuya; Maruyama, Chitose; Katano, Hajime

2014-08-01

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases.

PubMed

Miller, Bradley R; Sundlov, Jesse A; Drake, Eric J; Makin, Thomas A; Gulick, Andrew M

2014-10-01

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. © 2014 Wiley Periodicals, Inc.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

PubMed

Chen, Yaozong; Li, Tin Lok; Lin, Xingbang; Li, Xin; Li, Xiang David; Guo, Zhihong

2017-07-21

o -Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ling, Jiqiang; Peterson, Kaitlyn M.; Simonovic, Ivana

2014-03-12

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular ?? with a threonine (Thr) anticodon, MST1 also recognizes an unusual ??, which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employsmore » distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for ?? recognition, whereas the anticodon sequence is essential for aminoacylation of ??. The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate ??, reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix {alpha}11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.« less

Characterization of Acyl-CoA Synthetase Isoforms In Pancreatic Beta Cells: Gene Silencing Shows Participation of ACSL3 and ACSL4 In Insulin Secretion

PubMed Central

Ansari, Israr-ul H.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; O’Neill, Lucas M.; Zitur, Laura J.; Fernandez, Luis A.; Ntambi, James M.; MacDonald, Michael J.

2017-01-01

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ~ 50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion. PMID:28193492

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

PubMed

Jaremko, Matt J; Lee, D John; Patel, Ashay; Winslow, Victoria; Opella, Stanley J; McCammon, J Andrew; Burkart, Michael D

2017-10-10

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids.

PubMed

Beld, Joris; Abbriano, Raffaela; Finzel, Kara; Hildebrand, Mark; Burkart, Michael D

2016-04-01

In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes. We fed various odd and short chain fatty acids to the bacterium Escherichia coli, cyanobacterium Synechocystis sp. PCC 6803, green microalga Chlamydomonas reinhardtii and diatom Thalassiosira pseudonana. Major differences were observed, specifically in the ability among species to incorporate and elongate short chain fatty acids. We demonstrate that E. coli, C. reinhardtii, and T. pseudonana can produce longer fatty acid products from short chain precursors (C3 and C5), while Synechocystis sp. PCC 6803 lacks this ability. However, Synechocystis can incorporate and elongate longer chain fatty acids due to acyl-acyl carrier protein synthetase (AasS) activity, and knockout of this protein eliminates the ability to incorporate these fatty acids. In addition, expression of a characterized AasS from Vibrio harveyii confers a similar capability to E. coli. The ability to desaturate exogenously added fatty acids was only observed in Synechocystis and C. reinhardtii. We further probed fatty acid metabolism of these organisms by feeding desaturase inhibitors to test the specificity of long-chain fatty acid desaturases. In particular, supplementation with thia fatty acids can alter fatty acid profiles based on the location of the sulfur in the chain. We show that coupling sensitive gas chromatography mass spectrometry to supplementation of unnatural fatty acids can reveal major differences between fatty acid metabolism in various organisms. Often unnatural fatty acids have antibacterial or even therapeutic properties. Feeding of short

Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.

PubMed

Ben-Salem, Salma; Gleeson, Joseph G; Al-Shamsi, Aisha M; Islam, Barira; Hertecant, Jozef; Ali, Bassam R; Al-Gazali, Lihadh

2015-06-01

Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 6.3.5.4), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Effects of postprandial starvation on mRNA expression of endocrine-, amino acid and peptide transporter-, and metabolic enzyme-related genes in zebrafish (Danio rerio).