scholarly journals Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Leonardo Negron ◽  
Mark L. Patchett ◽  
Emily J. Parker
Keyword(s):  
Elevated Temperatures ◽  
Pyrococcus Furiosus ◽  
Shikimate Pathway ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Divalent Metal Ions ◽  
Biochemical Characterisation ◽  
Temperature Ranges ◽  
A Subunit ◽  
Dehydroquinate Synthase

Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30∘C followed by a heat treatment at 70∘C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and kcat 3.0 sec-1 at 60∘C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.

scholarly journals The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism

1998 ◽  
Vol 180 (24) ◽  
pp. 6668-6673 ◽  
Author(s):  
Chang-Jun Cha ◽  
Ronald B. Cain ◽  
Neil C. Bruce
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Sole Source ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Rhodococcus Rhodochrous ◽  
Natural Substrate ◽  
Ph Optimum ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native M r of 128,000, and a subunitM r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

scholarly journals 4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2

1991 ◽  
Vol 274 (3) ◽  
pp. 833-842 ◽  
Author(s):  
H H Locher ◽  
T Leisinger ◽  
A M Cook
Keyword(s):  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Comamonas Testosteroni ◽  
Visible Absorption ◽  
Mononuclear Iron ◽  
Purification And Properties ◽  
A Subunit ◽  
Protein Components

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

scholarly journals Kinetic characterization and thermostability of C. elegans cytoplasmic and mitochondrial malate dehydrogenases

2021 ◽  
Author(s):  
Matthew J. Thomas ◽  
Emma R. Cassidy ◽  
Devin S. Robinson ◽  
Katherine M. Walstrom
Keyword(s):  
Environmental Conditions ◽  
Elevated Temperatures ◽  
Homology Model ◽  
Anion Exchange Chromatography ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Salt Bridges ◽  
Recombinant Enzymes ◽  
C Elegans ◽  
Adverse Environmental Conditions

Malate dehydrogenase (MDH) catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate in the last step of the citric acid cycle. Eukaryotes have at least two MDH isozymes, one that is imported into the mitochondria and one that remains in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 (F46E10.10) and mitochondrial MDH-2 (F20H11.3) in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we determined that the KM values for oxaloacetate were 54 and 52 μM, and the KM values for NADH were 61 and 107 μM, for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH from a mixed population of worms and separated MDH-1 from MDH-2 using anion exchange chromatography. Both endogenous enzymes had a KM for oxaloacetate similar to that of the corresponding recombinant enzyme. The reaction velocities of the recombinant enzymes had slightly different temperature-dependencies: MDH-1 and MDH-2 had maximum activity at 40 °C and 35 °C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Molecular homology modeling predicted that MDH-1 had more salt-bridges between the subunits than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer ionic interaction between the subunits compared to mammalian MDH2 enzymes. These results suggest that the increased structural stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.

scholarly journals Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa

1992 ◽  
Vol 284 (2) ◽  
pp. 417-423 ◽  
Author(s):  
D J Clarke ◽  
S G George ◽  
B Burchell
Keyword(s):  
Mammalian Species ◽  
Liver Microsomes ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Apparent Homogeneity ◽  
Molecular Masses ◽  
A Subunit ◽  
Udp Glucuronosyltransferase ◽  
High Degree ◽  
Sepharose 4B

The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated 1-naphthol, but not bilirubin or steroids, and displayed a pI of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

Determination of fructooligosaccharides in milk powder using high performance anion-exchange chromatography coupled with pulsed amperometric detection

2014 ◽  
Vol 32 (12) ◽  
pp. 1380 ◽  
Author(s):  
Lijuan GENG ◽  
Junrong HUANG ◽  
Feng FENG ◽  
Pingping JIANG ◽  
Xiaogang CHU ◽  
...  
Keyword(s):  
Anion Exchange ◽  
High Performance ◽  
Milk Powder ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Pulsed Amperometric Detection

Correlation between protein desorption behavior and its adsorption enthalpy change in polymer grafted anion exchange chromatography

2021 ◽  
pp. 111853
Author(s):  
Joao Carlos Simoes-Cardoso ◽  
Nanako Hoshino ◽  
Yusuke Yoshimura ◽  
Chyi-Shin Chen ◽  
Cristina Dias-Cabral ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enthalpy Change ◽  
Adsorption Enthalpy
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