scholarly journals Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase

2021 ◽  
Vol 22 (8) ◽  
pp. 4231
Author(s):  
Giulia Murtas ◽  
Giorgia Letizia Marcone ◽  
Alessio Peracchi ◽  
Erika Zangelmi ◽  
Loredano Pollegioni
Keyword(s):  
Cancer Progression ◽  
Forward Direction ◽  
Biochemical Properties ◽  
Reverse Direction ◽  
Phosphate Binding ◽  
Biophysical Characterization ◽  
E Coli ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Metabolism

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

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

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.

Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt

2017 ◽  
Vol 1861 (11) ◽  
pp. 2702-2709 ◽  
Author(s):  
A. Gilardi ◽  
S.P. Bhamidimarri ◽  
M. Brönstrup ◽  
U. Bilitewski ◽  
R.K.R. Marreddy ◽  
...  
Keyword(s):  
Biophysical Characterization ◽  
E Coli

scholarly journals A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

2006 ◽  
Vol 72 (1) ◽  
pp. 245-251 ◽  
Author(s):  
Ju-Yuan Zhang ◽  
Jie Zou ◽  
Qiyu Bao ◽  
Wen-Li Chen ◽  
Li Wang ◽  
...  
Keyword(s):  
Arthrospira Platensis ◽  
Biochemical Properties ◽  
Salt Sensitivity ◽  
E Coli ◽  
Multiple Origins ◽  
Manic Depressive ◽  
Sodium Toxicity ◽  
Function Relationship ◽  
Eukaryotic Organisms

ABSTRACT 3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50] = 3.6 mM) but only slightly sensitive to Na+ (IC50 = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.

scholarly journals Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene (pdc) and Comparison to Bacterial Homologues

2002 ◽  
Vol 68 (6) ◽  
pp. 2869-2876 ◽  
Author(s):  
Krishnan Chandra Raj ◽  
Lee A. Talarico ◽  
Lonnie O. Ingram ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Codon Usage ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Biochemical Properties ◽  
Kinetic Properties ◽  
E Coli ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Zymobacter Palmae

ABSTRACT Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein−1) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

scholarly journals Characterization of Escherichia coliD-arabinose 5-phosphate isomerase encoded by kpsF: implications for group 2 capsule biosynthesis

2006 ◽  
Vol 395 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Timothy C. Meredith ◽  
Ronald W. Woodard
Keyword(s):  
Biochemical Properties ◽  
Coli Strain ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Catalytic Constant ◽  
Group 2 ◽  
Strain Cft073 ◽  
K Antigen ◽  
Pentose Pathway

In Escherichia coli, there are multiple paralogous copies of the enzyme API [A5P (D-arabinose 5-phosphate) isomerase], which catalyses the conversion of the pentose pathway intermediate Ru5P (D-ribulose 5-phosphate) into A5P. A5P is a precursor of Kdo (3-deoxy-D-manno-octulosonate), an integral carbohydrate component of various glycolipids coating the surface of the OM (outer membrane) of Gram-negative bacteria, including LPS (lipopolysaccharide) and many group 2 K-antigen capsules. The K-antigen-specific API KpsF has been cloned from the uropathogenic E. coli strain CFT073 and its biochemical properties characterized. Purified recombinant KpsF [K-API (K-antigen API)] is tetrameric and has optimal activity at pH 7.8. The enzyme is specific for A5P and Ru5P, with Km (app) values of 0.57 mM for A5P and 0.3 mM for Ru5P. The apparent kcat in the A5P to Ru5P direction is 15 and 19 s−1 in the Ru5P to A5P direction. While most of the properties are quite similar to its LPS API counterpart KdsD, the catalytic constant is nearly 10-fold lower. K-API is now the second Kdo biosynthetic related gene that has been characterized from the kps group 2 capsule cluster.

scholarly journals 5,10-Methylenetetrahydrofolate Reductase (MTHFR) Assay in the Forward Direction: Residual Activity in MTHFR Deficiency

2002 ◽  
Vol 48 (6) ◽  
pp. 835-843 ◽  
Author(s):  
Terttu Suormala ◽  
Gertraud Gamse ◽  
Brian Fowler
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Residual Activity ◽  
Forward Direction ◽  
Reverse Direction ◽  
Control Activity ◽  
Reliable Determination ◽  
Protein Assay ◽  
Control Value ◽  
Mthfr Deficiency

Abstract Background: Assay of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in homocysteine metabolism, is important for the study of severe and mild deficiency states. Because the conventional assay measures in the reverse direction, lacks sensitivity, and uses nonphysiologic substrates, the exact measurement and characterization of residual activity in easily accessible tissues have been difficult. Methods: To measure MTHFR in the physiologic direction, we determined the NADPH-dependent conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate by use of HPLC with fluorescence detection. Results: MTHFR activity in control fibroblast in the presence of FAD was maximal between pH 6.3 and 6.9, increased linearly up to 40 min and 80 μg protein/assay, and showed Kms of 30 μmol/L for NADPH and 26 μmol/L for 5,10-methylenetetrahydrofolate. Intraassay variation (CV) was 10%, interassay variation was 7.2%, and variation among 10 subcultures of the same cell line was 18%. Mean (SD) control activity was 431 (150) μU/mg protein (range, 242–910; n = 75), which is 2.5-fold higher than that with the reverse assay. After heat treatment (46 °C for 5 min), the activity showed a trimodal distribution corresponding to the 677TT (thermolabile; 15%), 677CT (35%), and 677CC (51%) genotypes. We found clearly measurable activity ranging from 2.6% to 25.6% of the mean control value in 15 patients with MTHFR deficiency, including 11 cell lines with zero activity in the reverse assay. Ten patients had complete enzyme deficiency. Conclusion: This assay allows reliable determination of residual activity in mutant fibroblasts and characterization of kinetic parameters for natural substrates.

scholarly journals Decoding sequence-level information to predict membrane protein expression

2017 ◽  
Author(s):  
Shyam M. Saladi ◽  
Nauman Javed ◽  
Axel Müller ◽  
William M. Clemons
Keyword(s):  
Sequence Information ◽  
Biophysical Characterization ◽  
E Coli ◽  
Membrane Protein Expression ◽  
Improve Model ◽  
Wide Range ◽  
Probability Of Success ◽  
Level Information ◽  
Major Bottleneck

SummaryThe expression of integral membrane proteins (IMPs) remains a major bottleneck in the characterization of this important protein class. IMP expression levels are currently unpredictable, which renders the pursuit of IMPs for structural and biophysical characterization challenging and inefficient. Experimental evidence demonstrates that changes within the nucleotide or amino-acid sequence for a given IMP can dramatically affect expression; yet these observations have not resulted in generalizable approaches to improved expression. Here, we develop a data-driven statistical predictor named IMProve, that, using only sequence information, increases the likelihood of selecting an IMP that expresses in E. coli. The IMProve model, trained on experimental data, combines a set of sequence-derived features resulting in an IMProve score, where higher values have a higher probability of success. The model is rigorously validated against a variety of independent datasets that contain a wide range of experimental outcomes from various IMP expression trials. The results demonstrate that use of the model can more than double the number of successfully expressed targets at any experimental scale. IMProve can immediately be used to identify favorable targets for characterization.

scholarly journals Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters

2020 ◽  
Author(s):  
Sunanda Mallick ◽  
Ashish Kumar ◽  
Hiren Dodia ◽  
Cyrus Alexander ◽  
Dileep Vasudevan ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Sequence Similarity ◽  
Direct Interaction ◽  
Biochemical Properties ◽  
Cytoplasmic Loop ◽  
Dynamic Component ◽  
E Coli

The peptidoglycan (PG) layer is an intricate and dynamic component of the bacterial cell wall, which requires a constant balance between its synthesis and hydrolysis. FtsEX complex present on the inner membrane is shown to transduce signals to induce PG hydrolysis. FtsE has sequence similarity with the nucleotide-binding domain (NBDs) of ABC transporters. The NBDs in most of the ABC transporters couple ATP hydrolysis to transport molecules inside or outside the cell. Also, this reaction cycle is driven by the dimerization of NBDs. Though extensive studies have been carried out on the E. coli FtsEX complex, it remains elusive regarding how FtsEX complex helps in signal transduction or transportation of molecules. Also, very little is known about the biochemical properties and ATPase activities of FtsE. Because of its strong interaction with the membrane-bound protein FtsX, FtsE stays insoluble upon overexpression in E. coli, and thus, most studies on E. coli FtsE in the past have used refolded FtsE. Here in this paper, for the first time, we report the soluble expression, purification, and biochemical characterization of FtsE from E. coli. The purified soluble FtsEexhibits high thermal stability, exhibits ATPase activity and has more than one ATP-binding site. We have also demonstrated a direct interaction between FtsEand the cytoplasmic loop of FtsX. Together, our findings suggest that during bacterial division, the ATPase cycle of FtsE and its interaction with the FtsX cytoplasmic loop may help to regulate the peptidoglycan hydrolysis at the mid cell.

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