Metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus: dynamic enzyme–enzyme interactions involved in the formation of the channelling complex

2004 ◽  
Vol 32 (2) ◽  
pp. 306-309 ◽  
Author(s):  
J. Massant ◽  
N. Glansdorff
Keyword(s):  
Protein Interactions ◽  
Pyrococcus Furiosus ◽  
Hyperthermophilic Archaea ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Carbamoyl Phosphate ◽  
Molecular Physiology ◽  
Metabolic Channelling ◽  
Aspartate Carbamoyltransferase ◽  
Carbamate Kinase

Protection of thermolabile metabolites and coenzymes is a somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles. Detailed information about the mechanisms used by thermophiles to protect these thermolabile metabolites and coenzymes is still scarce. A case in point is CP (carbamoyl phosphate), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. Recently we obtained the first evidence for a physical interaction between two hyperthermophilic enzymes for which kinetic evidence had suggested that these enzymes channel a highly thermolabile and potentially toxic intermediate. By physically interacting with each other, CKase (carbamate kinase) and OTCase (ornithine carbamoyltransferase) prevent thermodenaturation of CP in the aqueous cytoplasmic environment. The CP channelling complex involving CKase and OTCase or ATCase (aspartate carbamoyltransferase), identified in hyperthermophilic archaea, provides a good model system to investigate the mechanism of metabolic channelling and the molecular basis of protein–protein interactions in the physiology of extreme thermophiles.

scholarly journals New experimental approaches for investigating interactions betweenPyrococcus furiosuscarbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate

Archaea ◽  
2005 ◽  
Vol 1 (6) ◽  
pp. 365-373 ◽  
Author(s):  
Jan Massant ◽  
Nicolas Glansdorff
Keyword(s):  
Protein Interactions ◽  
Thermal Protection ◽  
Pyrococcus Furiosus ◽  
Titration Calorimetry ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Carbamoyl Phosphate ◽  
Molecular Physiology ◽  
Metabolic Channeling ◽  
Experimental Approaches

A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine carbamoyltransferase (OTC) fromPyrococcus furiosuswas provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein–protein interactions in the physiology of hyperthermophiles.

scholarly journals Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
James E. Hennessy ◽  
Melissa J. Latter ◽  
Somayeh Fazelinejad ◽  
Amy Philbrook ◽  
Daniel M. Bartkus ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Hyperthermophilic Archaea ◽  
High Yield ◽  
Water Runoff ◽  
High Catalytic Activity ◽  
Alkaline Ph ◽  
Biotechnological Applications ◽  
Carbamoyl Phosphate ◽  
Content Type ◽  
Carbamate Kinase

ABSTRACT Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus , the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus , the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis , and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate. IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

[21] Carbamoyl phosphate synthesis: Carbamate kinase from Pyrococcus furiosus

2001 ◽  
pp. 236-247 ◽  
Author(s):  
Matxalen Uriarte ◽  
Alberto Marina ◽  
Santiago Ramón-Maiques ◽  
Vicente Rubio ◽  
Virginie Durbecq ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Carbamoyl Phosphate ◽  
Carbamate Kinase

scholarly journals Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains.

1995 ◽  
Vol 15 (5) ◽  
pp. 2448-2456 ◽  
Author(s):  
M Crossley ◽  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Physical Interaction ◽  
Erythroid Cells ◽  
Protein Protein Interactions ◽  
Self Association ◽  
Zinc Finger Region ◽  
In Erythroid Cells

GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.

scholarly journals Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis

2012 ◽  
Vol 194 (18) ◽  
pp. 4941-4950 ◽  
Author(s):  
Melissa de Francesco ◽  
Jake Z. Jacobs ◽  
Filipa Nunes ◽  
Mónica Serrano ◽  
Peter T. McKenney ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Physical Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Protein Protein Interactions ◽  
Content Type ◽  
Morphogenetic Protein ◽  
Physical Interactions

ABSTRACTEndospore formation byBacillus subtilisis a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.

scholarly journals A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Wei Dong ◽  
Juan Lu ◽  
Xuejing Zhang ◽  
Yan Wu ◽  
Kaela Lettieri ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Protein Interactions ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cell Polarization ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Binding Domains ◽  
Electrostatic Binding

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein–protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

scholarly journals Detection of protein–protein interactions at the septin collar inSaccharomyces cerevisiaeusing a tripartite split-GFP system

2016 ◽  
Vol 27 (17) ◽  
pp. 2708-2725 ◽  
Author(s):  
Gregory C. Finnigan ◽  
Angela Duvalyan ◽  
Elizabeth N. Liao ◽  
Aspram Sargsyan ◽  
Jeremy Thorner
Keyword(s):  
Protein Interactions ◽  
Weak Interactions ◽  
Glycolytic Enzyme ◽  
Fluorescence Signal ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
New Information ◽  
Bud Neck ◽  
Molecular Ruler

Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein–protein interactions in a dynamic cytoskeletal structure—the septin collar at the yeast bud neck. We show, first, that for septin–septin interactions, this method yields a robust signal whose strength reflects the known spacing between the subunits in septin filaments and thus serves as a “molecular ruler.” Second, the method yields little or no spurious signal even with highly abundant cytosolic proteins readily accessible to the bud neck (including molecular chaperone Hsp82 and glycolytic enzyme Pgk1). Third, using two proteins (Bni5 and Hsl1) that have been shown by other means to bind directly to septins at the bud neck in vivo, we validate that the tripartite split-GFP method yields the same conclusions and further insights about specificity. Finally, we demonstrate the capacity of this approach to uncover additional new information by examining whether three other proteins reported to localize to the bud neck (Nis1, Bud4, and Hof1) are able to interact physically with any of the subunits in the septin collar and, if so, with which ones.

scholarly journals A comprehensive physical interaction map between the Turnip mosaic potyvirus and Arabidopsis thaliana proteomes

2021 ◽  
Author(s):  
Fernando Martínez ◽  
Christina Toft ◽  
Julia Hillung ◽  
Silvia Giménez-Santamarina ◽  
Lynne Yenush ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Interactions ◽  
Mode Of Action ◽  
Plant Viruses ◽  
Host Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Virus Protein ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Disease Etiology

Abstract Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Yet, the systems-level mode of action of plant viruses remains poorly understood. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel virus-host protein-protein interactions between Turnip mosaic virus (TuMV), a representative plant RNA virus, and Arabidopsis thaliana, one of its natural hosts. We found the RNA-dependent RNA polymerase NIb as the virus protein with the largest number of contacts. We verified a subset of 25 selected interactions in planta by bimolecular fluorescence complementation assays. We then constructed a comprehensive network comprising 399 TuMV-A. thaliana interactions to perform, together with intravirus and intrahost connections, detailed computational analyses. In particular, we found that the host proteins targeted by the virus participate in a higher number of infection-related functions, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. Overall, our results provide a comprehensive mechanistic description of a plant virus-host interplay, with potential impact on disease etiology, and reveal that plant and animal viruses share fundamental features in their mode of action.

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