Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2

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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Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

Microbiology ◽

10.1099/mic.0.051847-0 ◽

2011 ◽

Vol 157 (11) ◽

pp. 3059-3070 ◽

Cited By ~ 16

Author(s):

Hongchao Wang ◽

Bo Yang ◽

Guangfei Hao ◽

Yun Feng ◽

Haiqin Chen ◽

...

Keyword(s):

Biochemical Characterization ◽

De Novo ◽

Mortierella Alpina ◽

Recombinant Enzymes ◽

Homologous Proteins ◽

Genes Encoding ◽

Oleaginous Fungus ◽

Cell Processes ◽

Common Substrate

We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH4) in the lipid-producing fungus Mortierella alpina. The BH4 cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H2-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H2-NTP to 6-pyruvoyltetrahydropterin (PPH4), and sepiapterin reductase (SR) for the conversion of PPH4 to BH4, were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH4 in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH4 and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH4 biosynthesis pathway in a fungus.

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Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

Applied and Environmental Microbiology ◽

10.1128/aem.69.9.5423-5432.2003 ◽

2003 ◽

Vol 69 (9) ◽

pp. 5423-5432 ◽

Cited By ~ 23

Author(s):

Armelle Cochu ◽

Christian Vadeboncoeur ◽

Sylvain Moineau ◽

Michel Frenette

Keyword(s):

Biochemical Characterization ◽

Sugar Transport ◽

Streptococcus Thermophilus ◽

Phosphate Group ◽

Phosphotransferase System ◽

Whole Cells ◽

Genes Encoding ◽

Regulatory Functions

ABSTRACT In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABMan as well as membrane fragments containing IICMan and IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.

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Human- and Plant-Pathogenic Pseudomonas Species Produce Bacteriocins Exhibiting Colicin M-Like Hydrolase Activity towards Peptidoglycan Precursors

Journal of Bacteriology ◽

10.1128/jb.01824-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3657-3664 ◽

Cited By ~ 52

Author(s):

Hélène Barreteau ◽

Ahmed Bouhss ◽

Martine Fourgeaud ◽

Jean-Luc Mainardi ◽

Thierry Touzé ◽

...

Keyword(s):

Biochemical Characterization ◽

Hydrolase Activity ◽

New Class ◽

Low Concentrations ◽

Pseudomonas Species ◽

Genes Encoding ◽

Colicin M ◽

Undecaprenyl Phosphate

ABSTRACT Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg2+-dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.

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Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon

Microorganisms ◽

10.3390/microorganisms9112318 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2318

Author(s):

Dennis Winkler ◽

Sabrina Gfrerer ◽

Johannes Gescher

Keyword(s):

Isocitrate Dehydrogenase ◽

Reaction Rate ◽

Biochemical Characterization ◽

Divalent Cations ◽

Kinetic Studies ◽

Genetic System ◽

Binding Pocket ◽

Local Secondary Structure ◽

Genes Encoding

Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “Candidatus Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP+ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and kcat-values of 38.48 ± 1.62 and 43.99 ± 1.46 s−1 resulting in kcat/KM-values of 725 ± 107.62 and 22.69 ± 2.15 mM−1 s−1 for DL-isocitrate and NADP+, respectively. MhIDH’s exceptionally low affinity for NADP+, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP+ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.

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Identification and Characterization of trans-Isopentenyl Diphosphate Synthases Involved in Herbivory-Induced Volatile Terpene Formation in Populus trichocarpa

Molecules ◽

10.3390/molecules24132408 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2408 ◽

Cited By ~ 1

Author(s):

Nathalie D. Lackus ◽

Nora P. Petersen ◽

Raimund Nagel ◽

Axel Schmidt ◽

Sandra Irmisch ◽

...

Keyword(s):

Biochemical Characterization ◽

Populus Trichocarpa ◽

Insect Herbivory ◽

Isopentenyl Diphosphate ◽

Transcript Accumulation ◽

Sequence Comparisons ◽

Geranyl Diphosphate ◽

Genes Encoding

In response to insect herbivory, poplar releases a blend of volatiles that plays important roles in plant defense. Although the volatile bouquet is highly complex and comprises several classes of compounds, it is dominated by mono- and sesquiterpenes. The most common precursors for mono- and sesquiterpenes, geranyl diphosphate (GPP) and (E,E)-farnesyl diphosphate (FPP), respectively, are in general produced by homodimeric or heterodimeric trans-isopentenyl diphosphate synthases (trans-IDSs) that belong to the family of prenyltransferases. To understand the molecular basis of herbivory-induced terpene formation in poplar, we investigated the trans-IDS gene family in the western balsam poplar Populus trichocarpa. Sequence comparisons suggested that this species possesses a single FPP synthase gene (PtFPPS1) and four genes encoding two large subunits (PtGPPS1.LSU and PtGPPS2.LSU) and two small subunits (PtGPPS.SSU1 and PtGPPS.SSU2) of GPP synthases. Transcript accumulation of PtGPPS1.LSU and PtGPPS.SSU1 was significantly upregulated upon leaf herbivory, while the expression of PtFPPS1, PtGPPS2.LSU, and PtGPPS.SSU2 was not influenced by the herbivore treatment. Heterologous expression and biochemical characterization of recombinant PtFPPS1, PtGPPS1.LSU, and PtGPPS2.LSU confirmed their respective IDS activities. Recombinant PtGPPS.SSU1 and PtGPPS.SSU2, however, had no enzymatic activity on their own, but PtGPPS.SSU1 enhanced the GPP synthase activities of PtGPPS1.LSU and PtGPPS2.LSU in vitro. Altogether, our data suggest that PtGPPS1.LSU and PtGPPS2.LSU in combination with PtGPPS.SSU1 may provide the substrate for herbivory-induced monoterpene formation in P. trichocarpa. The sole FPP synthase PtFPPS1 likely produces FPP for both primary and specialized metabolism in this plant species.

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Identification and biochemical characterization of two novel peroxiredoxins in a liver fluke,Clonorchis sinensis

Parasitology ◽

10.1017/s0031182011000813 ◽

2011 ◽

Vol 138 (9) ◽

pp. 1143-1153 ◽

Cited By ~ 9

Author(s):

Y.-A. BAE ◽

S.-H. KIM ◽

E.-G. LEE ◽

W.-M. SOHN ◽

Y. KONG

Keyword(s):

Biochemical Characterization ◽

Expression Patterns ◽

Clonorchis Sinensis ◽

Type Equation ◽

Protein Product ◽

Steady State Kinetic ◽

Genes Encoding ◽

Secretory Products ◽

Almost All

SUMMARYWe identified 2 novel genes encoding different 2-Cys peroxiredoxins (PRxs), designated CsPRx2 and CsPRx3, inClonorchis sinensis, which invades the human hepatobiliary tracts. TheCsPRx2gene expression was temporally increased along with the parasite's development and its protein product was detected in almost all parts of adult worms including subtegument, as well as excretory-secretory products. Conversely,CsPRx3expression was temporally maintained at a basal level and largely restricted within interior parts of various tissues/organs. The recombinant forms of CsPRx proteins exhibited reducing activity against various hydroperoxides in the presence of either thioredoxin or glutathione (GSH) as a reducing equivalent, although they preferred H2O2and GSH as a catalytic substrate and electron donor, respectively. A steady-state kinetic study demonstrated that the CsPRx proteins followed a saturable, Michaelis-Menten-type equation with the catalytic efficiencies (kcat/Km) ranging from 103to 104M−1s−1, somewhat lower than those for other PRxs studied (104–105M−1s−1). The expression patterns and histological distributions specific to CsPRx2 and CsPRx3 might suggest different physiological functions of the antioxidant enzymes in protecting the worms against oxidative damage.

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A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

mBio ◽

10.1128/mbio.02285-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 38

Author(s):

Zhen Yan ◽

Mingyu Wang ◽

James G. Ferry

Keyword(s):

Electron Transfer ◽

Electron Transport ◽

Biochemical Characterization ◽

Fundamental Principle ◽

Methanosarcina Acetivorans ◽

Transport Pathway ◽

Heterodisulfide Reductase ◽

Genes Encoding ◽

The Individual

ABSTRACT Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2 -reducing methanogenic anaerobes (methanogens) from the domain Archaea . Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea . The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli , and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420 (F 420 H 2 ) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420 H 2 and reduction of ferredoxin with the exergonic oxidation of F 420 H 2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. IMPORTANCE Discovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2 -reducing methanogens to include diverse prokaryotes from the domains Bacteria and Archaea . The unprecedented coenzyme F 420 -dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4 -oxidizing pathways. The results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.

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Biochemical adaptations of two sugar kinases from the hyperthermophilic archaeon Pyrococcus furiosus

Biochemical Journal ◽

10.1042/bj20011597 ◽

2002 ◽

Vol 366 (1) ◽

pp. 121-127 ◽

Cited By ~ 39

Author(s):

Corné H. VERHEES ◽

Denise G.M. KOOT ◽

Thijs J.G. ETTEMA ◽

Cor DIJKEMA ◽

Willem M. de VOS ◽

...

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Pyrococcus Furiosus ◽

Phosphoryl Group ◽

Hyperthermophilic Archaeon ◽

High Affinity ◽

Optimal Activity ◽

Genes Encoding ◽

Modified Embden Meyerhof Pathway

The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden—Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase. In the present study, we report the characterization of a P. furiosus galactokinase (GALK) and its comparison with the P. furiosus ADP-GLK. The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity. Both enzymes are specific kinases with an optimal activity at approx. 90°C. Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP. There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P. furiosus.

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