Purification and characterization of arginase from Neurospora crassa.

Borkovich, Katherine A.; Weiss, Richard L.

doi:10.1016/s0021-9258(18)48206-3

“…The fungal mycelia were collected by filtration, the pellets were washed with sterile distilled water, and their intracellular crude proteins were extracted [ 29 , 30 ]. Five grams of each fresh fungal biomass were grinded into fine powder in liquid nitrogen, then dispensed to 5 ml Tris-HCl buffer (pH 7.0, 50 mM) with 1 mM phenylmethansulphonyl floride (PMSF), 10 μl β- mercaptoethanol and 1 mM CaCl 2 [ 42 – 44 ]. The mixture was shaken for 10 min by vortex and centrifuged at 8000 rpm for 10 min at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…The deamination activity of CDA was assayed as described by Sakai et al [ 44 , 45 ] with slight modifications. The reaction mixture contains 100 mM cytosine and 0.5 ml of enzyme in Tris-HCl buffer (pH 7.0, 50 mM) in 1 ml total volume.…”

Section: Methodsmentioning

confidence: 99%

Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity

El-Sayed

¹

,

Rady

²

,

Mohamed

³

et al. 2023

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Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-flurocytosine into 5-flurouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efficiency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screening for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with affordable stability and catalytic efficiency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purified, with purification 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purified CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH 7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of effect of dextran conjugation on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The affinity of CDA-conjugates (Km 0.15 mM) and free CDA (Km 0.22 mM) to deaminate 5-fluorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the different cell lines “MDA-MB, HepG-2, and PC-3” was significantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.

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Arginase

Bewley

¹

,

Flanagan

²

2004

Handbook of Metalloproteins

0

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Arginase is a manganese hydrolase related to agmatinases, formiminoglutamases, and proclavaminate amidino hydrolase. It hydrolyzes L‐arginine to L‐ornithine and urea and occurs in bacteria, yeast, plants, invertebrates, and vertebrates. In mammalian species cytosolic type I and mitochondrial type II arginase are found. Type I arginases, located in the liver, catalyze the final step in the urea cycle. Type II arginases are extrahepatic and catalyze the same reaction as type I arginases, but due to their cellular location, give rise to different biological functions that center on regulating the levels of L‐arginine or L‐ornithine. For example, they are thought to regulate the concentrations of L‐arginine, a biosynthetic precursor of nitric oxide (NO), and thus, participate in the NO signaling pathway. By controlling the concentration of L‐ornithine, they affect the biosynthesis pathway of glutamate, proline, and polyamines. Arginase monomers have molecular weights of about 30 to 40 kDa and generally function as homo‐oligomers. All spectroscopic measurements have been made using the rat liver arginase and the Mn(II) EPR spectra are typical of μ‐ligand bridge spin‐coupled Mn 2 (II,II) centers. Crystal structures of rat and Bacillus caldovelox arginases have been determined. Rat liver arginase is a homotrimer. Each monomer of 323 amino acids has a globular shape and comprises an eight‐stranded parallel ß‐sheet packed on either side by helices to form a single α/ß domain. Each monomer contains its own active site. Two catalytic manganese ions, Mn A and Mn B are located at the bottom of the active site. Mn A is coordinated by a histidine, three aspartates, and two water molecules. Mn B is also coordinated by a histidine and three aspartates but by one water molecule only. One of the aspartates is bidentately coordinated (Mn B ), two others bridge both Mn ions, one with one side chain oxygen to both Mn ions, the other one with each side chain oxygen individually to each Mn ion. Both Mn ions display an octahedral coordination. The B. caldovelox arginase is a homohexamer with 32 point group symmetry. Each monomer contains 299 amino acid residues. Overall fold and active site geometry is very similar to that of the rat enzyme. Crystal structures of arginases in complex with L‐arginine, L‐lysine, L‐ornithine, and 2( S )‐amino‐6‐borohexanoic acid (ABH) have been determined and have helped, together with kinetic studies, to formulate a structure‐based catalytic mechanism for arginase in parallel to an EPR‐based mechanism.

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Arginase

Bewley

¹

,

Flanagan

²

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

1

0

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Arginase is a manganese hydrolase related to agmatinases, formiminoglutamases, and proclavaminate amidino hydrolase. It hydrolyzes L‐arginine to L‐ornithine and urea and occurs in bacteria, yeast, plants, invertebrates, and vertebrates. In mammalian species cytosolic type I and mitochondrial type II arginase are found. Type I arginases, located in the liver, catalyze the final step in the urea cycle. Type II arginases are extrahepatic and catalyze the same reaction as type I arginases, but due to their cellular location, give rise to different biological functions that center on regulating the levels of L‐arginine or L‐ornithine. For example, they are thought to regulate the concentrations of L‐arginine, a biosynthetic precursor of nitric oxide (NO), and thus, participate in the NO signaling pathway. By controlling the concentration of L‐ornithine, they affect the biosynthesis pathway of glutamate, proline, and polyamines. Arginase monomers have molecular weights of about 30 to 40 kDa and generally function as homo‐oligomers. All spectroscopic measurements have been made using the rat liver arginase and the Mn(II) EPR spectra are typical of μ‐ligand bridge spin‐coupled Mn 2 (II,II) centers. Crystal structures of rat and Bacillus caldovelox arginases have been determined. Rat liver arginase is a homotrimer. Each monomer of 323 amino acids has a globular shape and comprises an eight‐stranded parallel ß‐sheet packed on either side by helices to form a single α/ß domain. Each monomer contains its own active site. Two catalytic manganese ions, Mn A and Mn B are located at the bottom of the active site. Mn A is coordinated by a histidine, three aspartates, and two water molecules. Mn B is also coordinated by a histidine and three aspartates but by one water molecule only. One of the aspartates is bidentately coordinated (Mn B ), two others bridge both Mn ions, one with one side chain oxygen to both Mn ions, the other one with each side chain oxygen individually to each Mn ion. Both Mn ions display an octahedral coordination. The B. caldovelox arginase is a homohexamer with 32 point group symmetry. Each monomer contains 299 amino acid residues. Overall fold and active site geometry is very similar to that of the rat enzyme. Crystal structures of arginases in complex with L‐arginine, L‐lysine, L‐ornithine, and 2( S )‐amino‐6‐borohexanoic acid (ABH) have been determined and have helped, together with kinetic studies, to formulate a structure‐based catalytic mechanism for arginase in parallel to an EPR‐based mechanism.

show abstract

Purification and characterization of arginase from Neurospora crassa.

Cited by 43 publications

References 36 publications

Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity

Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity

Arginase

Arginase

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