A novel architecture of 600 kDa bacterial citrate lyase complex

Citrate lyase activity exists in eukaryotes, bacteria, and archaea and plays a central role in cellular energy metabolism. There are two types of citrate lyases, ATP-dependent and ATP-independent. The latter, which exists only in bacteria, converts citrate to acetate and oxaloacetate using a two-step reaction catalyzed by a stable heterotrimeric protein complex comprising alpha-beta-gamma subunits. We selected three bacterial genomes in which the alpha-beta-gamma subunits of citrate lyase are all encoded on the chromosome in a continuous, co-cistronic geometry. The genes encoding the entire complex were subsequently amplified together using a single PCR reaction on genomic DNA. This approach enabled the entire citrate lyase complex from three organisms to be purified to homogeneity using a single step of Ni-NTA chromatography followed by gel-filtration chromatography. One of the three citrate lyase complexes purified yielded crystals diffracting to 4 Å. Six-fold non-crystallographic symmetry averaging enabled a high quality structure to be determined for the 18-subunit alpha-beta-gamma citrate lyase complex. This structure provides insight into the multistep catalytic mechanism employed by this enzyme.

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The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin

Biochemical Journal ◽

10.1042/bj2680393 ◽

1990 ◽

Vol 268 (2) ◽

pp. 393-399 ◽

Cited By ~ 7

Author(s):

H K Paudel ◽

G M Carlson

Keyword(s):

Quaternary Structure ◽

Gel Filtration ◽

Phosphorylase Kinase ◽

Gamma Delta ◽

Gel Filtration Chromatography ◽

Structural Role ◽

Low Concentrations ◽

Gamma Subunits ◽

Alpha Beta ◽

Delta Subunit

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, β- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, β- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only β-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.

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The role of heterotrimeric G proteins in the control of symbiosis development in legume plants

BIO Web of Conferences ◽

10.1051/bioconf/20202303004 ◽

2020 ◽

Vol 23 ◽

pp. 03004

Author(s):

Andrey D. Bovin ◽

Irina V. Leppyanen ◽

Olga A. Pavlova ◽

Elena A. Dolgikh

Keyword(s):

Gene Expression ◽

G Proteins ◽

Heterotrimeric G Proteins ◽

Subunit Gene ◽

Gene Suppression ◽

Gamma Subunits ◽

Genes Encoding ◽

Specific Proteins ◽

Alpha Beta

Heterotrimeric G proteins are involved in the regulation of signaling pathways in eukaryotes. Previously, the data about possible participation of heterotrimeric G proteins in the regulation of nodulation in legumes were obtained, however, specific proteins, their composition and role in this process remain poorly understood. In this work searching of the genes encoding the alpha, beta, and gamma subunits of heterotrimeric G proteins based on an analysis of the Pisum sativum L. genome was performed, as well as the dynamics of the gene expression encoding the particular subunits of G proteins in the process of symbiosis was studied. In addition, a significant effect of beta 1-subunit gene suppression by RNA interference on the nodulation process was revealed.

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Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism

Journal of Virology ◽

10.1128/jvi.01784-07 ◽

2007 ◽

Vol 82 (6) ◽

pp. 2620-2630 ◽

Cited By ~ 22

Author(s):

Mulu Z. Tesfay ◽

Jun Yin ◽

Christina L. Gardner ◽

Mikhail V. Khoretonenko ◽

Nadejda L. Korneeva ◽

...

Keyword(s):

Sindbis Virus ◽

Antiviral Effect ◽

Protein Accumulation ◽

Rnase L ◽

80S Ribosome ◽

Host Protection ◽

Beta Interferon ◽

Genes Encoding ◽

Alpha Beta ◽

Antiviral Proteins

ABSTRACT The alpha/beta interferon (IFN-α/β) response is critical for host protection against disseminated replication of many viruses, primarily due to the transcriptional upregulation of genes encoding antiviral proteins. Previously, we determined that infection of mice with Sindbis virus (SB) could be converted from asymptomatic to rapidly fatal by elimination of this response (K. D. Ryman et al., J. Virol. 74:3366-3378, 2000). Probing of the specific antiviral proteins important for IFN-mediated control of virus replication indicated that the double-stranded RNA-dependent protein kinase, PKR, exerted some early antiviral effects prior to IFN-α/β signaling; however, the ability of IFN-α/β to inhibit SB and protect mice from clinical disease was essentially undiminished in the absence of PKR, RNase L, and Mx proteins (K. D. Ryman et al., Viral Immunol. 15:53-76, 2002). One characteristic of the PKR/RNase L/Mx-independent antiviral effect was a blockage of viral protein accumulation early after infection (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). We show here that IFN-α/β priming induces a PKR-independent activity that inhibits m7G cap-dependent translation at a step after association of cap-binding factors and the small ribosome subunit but before formation of the 80S ribosome. Furthermore, the activity targets mRNAs that enter across the cytoplasmic membrane, but nucleus-transcribed RNAs are relatively unaffected. Therefore, this IFN-α/β-induced antiviral activity represents a mechanism through which IFN-α/β-exposed cells are defended against viruses that enter the cytoplasm, while preserving essential host activities, including the expression of antiviral and stress-responsive genes.

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Four structurally distinct, non-DNA-binding subunits of human nuclear respiratory factor 2 share a conserved transcriptional activation domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.102 ◽

1995 ◽

Vol 15 (1) ◽

pp. 102-111 ◽

Cited By ~ 86

Author(s):

S Gugneja ◽

J V Virbasius ◽

R C Scarpulla

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Transcriptional Activation ◽

Deletion Mapping ◽

Rnase Protection ◽

Nuclear Respiratory Factor ◽

Gamma Subunits ◽

Nuclear Respiratory Factor 2 ◽

Alpha Beta ◽

Beta 2

Nuclear respiratory factor 2 (NRF-2) was previously purified to near homogeneity from HeLa cells on the basis of its ability to bind tandem recognition sites in the rat cytochrome oxidase subunit IV (RCO4) promoter. It consisted of five subunits, alpha, beta 1, beta 2, gamma 1, and gamma 2. Sequencing of tryptic peptides from alpha and from mixtures of the two beta or two gamma subunits revealed sequence identities with subunits of the mouse GA-binding protein (GABP), a ubiquitously expressed ETS domain activator composed of three subunits, alpha, beta 1, and beta 2. To understand the precise relationship between NRF-2 and GABP, cDNAs for all five NRF-2 subunits have now been cloned and their products have been overexpressed. The results establish that the two additional NRF-2 subunits are molecular variants that differ from GABP beta 1 and beta 2 by having a 12-amino-acid insertion containing two serine doublets. PCR and RNase protection assays show that mRNAs for these variants are expressed in the human but not the rodent cells and tissues examined. The insertion did not alter the ability of the beta and gamma subunits to associate with alpha, the DNA-binding subunit, nor did it affect the ability of NRF-2 beta 1 or beta 2 to direct high-affinity binding of alpha to tandem sites in the RCO4 promoter. In addition, the four NRF-2 beta and gamma subunits were equally proficient in activating transcription in transfected cells when fused to a GAL4 DNA-binding domain. The domain responsible for this transcriptional activation was localized by deletion mapping to a region of approximately 70 amino acids that is conserved in all four NRF-2 beta and gamma subunits. The repeated glutamine-containing hydrophobic clusters within this region bear a strong resemblance to those recently implicated in protein-protein interactions within the transcriptional apparatus.

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Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

10.1101/303594 ◽

2018 ◽

Author(s):

Krithika Rajagopalan ◽

Jonathan Dworkin

Keyword(s):

Biochemical Characterization ◽

Regulatory Protein ◽

Biochemical Properties ◽

Bacterial Genomes ◽

Phosphatase Inhibitors ◽

E Coli ◽

Specificity And Sensitivity ◽

Genes Encoding ◽

Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

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High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

Plants ◽

10.3390/plants9030304 ◽

2020 ◽

Vol 9 (3) ◽

pp. 304 ◽

Cited By ~ 1

Author(s):

Alexander Hofmann ◽

Sophia Müller ◽

Thomas Drechsler ◽

Mareike Berleth ◽

Katharina Caesar ◽

...

Keyword(s):

Osmotic Stress ◽

Stress Responses ◽

Histidine Kinase ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Cd Spectroscopy ◽

Single Step ◽

Signaling Mechanism ◽

Wide Range

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.

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Essential carboxy groups in xylanase A

Biochemical Journal ◽

10.1042/bj2700091 ◽

1990 ◽

Vol 270 (1) ◽

pp. 91-96 ◽

Cited By ~ 25

Author(s):

M R Bray ◽

A J Clarke

Keyword(s):

Catalytic Mechanism ◽

Ph Dependence ◽

Schizophyllum Commune ◽

Gel Filtration ◽

Enzymic Activity ◽

Water Soluble ◽

Significant Activity ◽

Nitrobenzoic Acid ◽

Woodward’S Reagent K ◽

Activity Loss

An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.

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The effect of the γ-subunit of the cyclic GMP phosphodiesterase of bovine and frog (Rana catesbiana) retinal rod outer segments on the kinetic parameters of the enzyme

Biochemical Journal ◽

10.1042/bj2650655 ◽

1990 ◽

Vol 265 (3) ◽

pp. 655-658 ◽

Cited By ~ 22

Author(s):

M M Whalen ◽

M W Bitensky ◽

D J Takemoto

Keyword(s):

Catalytic Activity ◽

Cyclic Gmp ◽

Active Sites ◽

Outer Segment ◽

Maximal Activity ◽

Inhibitory Effect ◽

Gamma Subunit ◽

Rod Outer Segment ◽

Gamma Subunits ◽

Alpha Beta

Rod-outer-segment cyclic GMP phosphodiesterase (PDE) (subunit composition alpha beta gamma 2) contains catalytic activity in alpha beta. The gamma-subunits are inhibitors. Removal of the gamma-subunits increases Vmax. without affecting the Km. The inhibitory effect of a single gamma-subunit (alpha beta gamma) on the Vmax. of alpha beta is much greater in bovine than in frog (Rana catesbiana) PDE. Bovine PDE in the alpha beta gamma 2 state has a Vmax. that is 2.6 +/- 0.4% of the Vmax. of alpha beta. The removal of one gamma-subunit to give alpha beta gamma results in a Vmax. 5.2 +/- 1% of that for maximal activity. Frog alpha beta gamma 2 has a Vmax. 10.8 +/- 2%, and alpha beta gamma has a Vmax. 50 +/- 18%, of the Vmax. of alpha beta. These data suggest that a single gamma-subunit can inhibit the catalytic activity of active sites on both alpha- and beta-subunits in bovine, but not in frog, rod-outer-segment PDE.

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