The cloning, expression and crystallisation of a thermostable arginase

Bewley, Maria C.; Lott, J.S.; Baker, Edward N.; Patchett, Mark L.

doi:10.1016/0014-5793(96)00459-0

Cited by 26 publications

(8 citation statements)

References 29 publications

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“…The addition of Mn 2+ increased enzyme activity by eightfold. This result agreed well with results from most organisms, such as human liver , B. caldovelox , and B. thuringiensis . It is reported that the active site of l ‐arginase contains two Mn 2+ ions (Mn 2+ A and Mn 2+ B ), which are coordinated by Asp and His residues .…”

Section: Resultssupporting

confidence: 90%

“…The alignment was performed using DNAMAN 7 program. G. thermodenitrificans arginase had the highest homologous (95.6% identity) and the lowest homologous (22.6% identity) in amino acid sequence with arginase from Bacillus caldovelox DSM411 and Helicobacter pylori B8 , respectively. In addition, ABO65532 exhibited 73.6%, and 69.0% amino acid sequence similarities with arginase from Bacillus anthracis BFV and B. subtilis 168 , respectively.…”

Section: Resultsmentioning

confidence: 99%

“…The effects of buffers were also determined, and showed that glycine–NaOH buffer was superior to the other buffers. The optimal pH for other l ‐arginases, such as B. thuringiensis is pH 10.0, S. cerevisiae is pH 9.5, Bacillus brevis and B. caldovelox are pH 9.0, and human liver is pH 8.0. However, the maximal activity of H. pylori is at pH 6.0.…”

Section: Resultsmentioning

confidence: 99%

“…A high optimum temperature is beneficial for enhancing reaction velocity, increasing the solubility of substrate and product, and reducing microbial contamination . However, temperatures higher than 80 °C may inactivate the enzyme and reduce l ‐ornithine production .…”

Section: Resultsmentioning

confidence: 99%

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Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Huang

Zhang

et al. 2015

Biotech and App Biochem

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Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l-ornithine from l-arginine. In this article, the l-arginase gene encoding the Geobacillus thermodenitrificans NG80-2 was cloned and overexpressed in Escherichia coli. The specific activity of the purified enzyme was 138.3 U/mg. The molecular mass of the l-arginase was approximately 33.0 kDa as estimated by SDS-PAGE and 192.0 kDa as determined by gel-filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg(2+) , Cu(2+) , Ba(2+) , Ca(2+) , and Zn(2+) . Activity was optimal at pH 9.0 and 80 °C, and the protein was stable at 40 and 50 °C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis-Menten constant (Km ) and catalytic efficiency (kcat /Km ) were measured to be 171.9 mM and 3.8 mM(-1) s(-1) , respectively. Trp and His residues were directly involved in the l-arginase activity evaluated by inactivation agents. The biosynthesis yield of l-ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Huang

Zhang

et al. 2015

Biotech and App Biochem

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“…The metal cluster is located in a 15 Å deep cleft with the Mn 2+ atoms 3.3 Å apart and bridged by a solvent molecule [35]. Structures from the bacteria Bacillus caldovelox [36,37], human arginase I [38] and II [39], rat arginase I [35,40] and Thermus thermophilus [Protein Data Bank (PDB) codes: 2EF4, 2EF5 and 2EIV] have been determined. In the mechanism proposed by Kanyo et al.…”

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confidence: 99%

The activity of Plasmodium falciparum arginase is mediated by a novel inter‐monomer salt‐bridge between Glu295–Arg404

Wells¹,

Müller²,

Wrenger³

et al. 2009

The FEBS Journal

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The polyamines putrescine, spermine and spermidine are near ubiquitous polycationic aliphatic amines required for a number of essential cellular processes, particularly in organisms undergoing rapid proliferation [1][2][3]. These processes involve the stabilization of macromolecules [4][5][6] and progression through the cell cycle [7]. Additionally, certain secondary metabolites, such as the post-translationally modified amino acid, hypusine [1,8], and the glutathione analogue, trypanothione, in Trypanosoma [9], require polyamines for their biosynthesis. Polyamine biosynthesis has been identified as a possible therapeutic target for various parasitic diseases [10,11], cancers [12] and even HIV via the requirement for hypusine [13]. Putrescine is synthesized by the decarboxylation of ornithine (ornithine decarboxylase) and serves as substrate for the addition of aminopropyl groups to form spermidine and spermine. The aminopropyl groups are donated A recent study implicated a role for Plasmodium falciparum arginase in the systemic depletion of arginine levels, which in turn has been associated with human cerebral malaria pathogenesis. Arginase (EC 3.5.3.1) is a multimeric metallo-protein that catalyses the hydrolysis of arginine to ornithine and urea by means of a binuclear spin-coupled Mn 2+ cluster in the active site. A previous report indicated that P. falciparum arginase has a strong dependency between trimer formation, enzyme activity and metal co-ordination. Mutations that abolished Mn 2+ binding also caused dissociation of the trimer; conversely, mutations that abolished trimer formation resulted in inactive monomers. By contrast, the monomers of mammalian (and therefore host) arginase are also active. P. falciparum arginase thus appears to be an obligate trimer and interfering with trimer formation may therefore serve as an alternative route to enzyme inhibition. In the present study, the mechanism of the metal dependency was explored by means of homology modelling and molecular dynamics. When the active site metals are removed, loss of structural integrity is observed. This is reflected by a larger equilibration rmsd for the protein when the active site metal is removed and some loss of secondary structure. Furthermore, modelling revealed the existence of a novel inter-monomer salt-bridge between Glu295 and Arg404, which was shown to be associated with the metal dependency. Mutational studies not only confirmed the importance of this salt-bridge in trimer formation, but also provided evidence for the independence of P. falciparum arginase activity on trimer formation.Abbreviations NP, constant number of atoms and constant pressure; NPT, constant number of atoms, constant pressure and temperature; PfArg, Plasmodium falciparum arginase.

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Arginase

Bewley

Flanagan

2004

Handbook of Metalloproteins

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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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The cloning, expression and crystallisation of a thermostable arginase

Cited by 26 publications

References 29 publications

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

The activity of Plasmodium falciparum arginase is mediated by a novel inter‐monomer salt‐bridge between Glu295–Arg404

Arginase

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