Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.1 (asparaginase)
2,695 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial L-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia for over 30 y. However, their use is limited owing to the glutaminase activity of the administered enzymes, which results in serious side effects. In contrast, L-asparaginase from Erwinia carotovora exhibits low glutaminase activity at physiological concentrations of L-asparagine and L-glutamine in the blood. Recombinant Er. carotovora L-asparaginase was crystallized in the presence of L-glutamate by the hanging-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 16-18%(w/v) PEG 3350 and 0.2 M NaF. X-ray diffraction data were collected to 2.6 A at 293 K using an in-house rotating-anode generator. The crystals belong to the monoclinic P2(1) space group, with unit-cell parameters a = 78.0, b = 112.3, c = 78.7 A, beta = 101.9 degrees and a homotetramer in the crystallographic asymmetric unit. A molecular-replacement solution has been found and refinement is currently in progress. The crystal structure may provide leads towards protein-engineering efforts aimed at safer asparaginase administration in leukaemia treatment.
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PMID:Crystallization and preliminary crystallographic analysis of L-asparaginase from Erwinia carotovora. 1651 Oct 54

Bacterial L-asparaginases catalyzing hydrolysis of L-asparagine up to L-aspartate and ammonia, are used in medical practice for treatment of acute lymphoblastic leukemia. The long-term therapy with these preparations is accompanied by a number of side effects, which are attributed to glutaminase activity of L-asparaginase. Substrate specificity and activity of L-asparaginases are directly associated with the process of enzyme oligomerization. It is active only in the tetrameric form as the active sites are located in contact areas between monomers. The present work is devoted to homology modeling of spatial structure of L-asparaginase from Erwinia carotovora, the comparative molecular-graphic analysis of subunits interfaces, as well as development of experimental approach for enzyme oligomerization study. L-asparaginase was immobilized on a CM5 chip surface of optical biosensor Biacore 3000 based on the surface plasmon resonance technology. The dissociation process of enzyme tetrameric complexes up to monomers and subsequent oligomerization process have been registered.
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PMID:[Oligomerization of L-asparaginase from Erwinia carotovora]. 1689 84

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.
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PMID:Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase. 1693 16

Pseudomonas putida KT2440 metabolizes a wide range of carbon and nitrogen sources, including many amino acids. In this study, a sigma54-dependent two-component system that controls the uptake and metabolism of acidic amino acids was identified. The system (designated aau, for acidic amino acid utilization) involves a sensor histidine kinase, AauS, encoded by PP1067, and a response regulator, AauR, encoded by PP1066. aauR and aauS deletion mutants were unable to efficiently utilize aspartate (Asp), glutamate (Glu), and glutamine (Gln) as sole sources of carbon and nitrogen. Growth of the mutants was partially restored when the above-mentioned amino acids were supplemented with glucose or succinate as an additional carbon source. Uptake of Gln, Asp, and asparagine (Asn) by the aauR mutant was moderately reduced, while Glu uptake was severely impaired. In the absence of glucose, the aauR mutant even secreted Glu into the medium. Furthermore, disruption of aauR affected the activities of several key enzymes of Glu and Asp metabolism, leading to the intracellular accumulation of Glu and greatly reduced survival times under conditions of nitrogen starvation. By a proteomics approach, four major proteins were identified that are downregulated during growth of the aauR mutant on Glu. Two of these were identified as periplasmic glutaminase/asparaginase and the solute-binding protein of a Glu/Asp transporter. Transcriptional analysis of lacZ fusions containing the putative promoter regions of these genes confirmed that their expression is indeed affected by the aau system. Three further periplasmic solute-binding proteins were strongly expressed during growth of the aauR deletion mutant on Glu but downregulated during cultivation on glucose/NH4+. These systems may be involved in amino acid efflux.
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PMID:The AauR-AauS two-component system regulates uptake and metabolism of acidic amino acids in Pseudomonas putida. 1702 Dec 7

Structural-based mutational analysis of salt-tolerant glutaminase from Micrococcus luteus K-3 (Micrococcus glutaminase) revealed that three amino acid residues, S64, K67, and E160, were essential to a catalytic reaction. The result suggested that Micrococcus glutaminase had a possible catalytic mechanism similar to class A beta-lactamase rather than glutaminase-asparaginase from Pseudomonas 7A.
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PMID:Analysis of essential amino acid residues for catalytic activity of glutaminase from Micrococcus luteus K-3. 1711 85

We describe an ATP-binding cassette (ABC) transporter in Pseudomonas putida KT2440 that mediates the uptake of glutamate and aspartate. The system (AatJMQP, for acidic amino acid transport) is encoded by an operon involving genes PP1071-PP1068. A deletion mutant with inactivated solute-binding protein (KTaatJ) failed to grow on Glu and Gln as sole sources of carbon and nitrogen, while a mutant lacking a functional nucleotide-binding domain (KTaatP) was able to adapt to growth on Glu after an extended lag phase. Uptake of Glu and Asp by either mutant was greatly impaired at both low and high amino acid concentrations. The purified solute-binding protein AatJ exhibited high affinity towards Glu and Asp (K(d)=0.4 and 1.3 muM, respectively), while Gln and Asn as well as dicarboxylates (succinate and fumarate) were bound with much lower affinity. We further show that the expression of AatJMQP is controlled by the sigma(54)-dependent two-component system AauRS. Binding of the response regulator AauR to the aat promoter was examined by gel mobility shift assays and DNase I footprinting. By in silico screening, the AauR-binding motif (the inverted repeat TTCGGNNNNCCGAA) was detected in further P. putida KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the H(+)/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.
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PMID:Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system. 1831 26

Bacterial L-asparaginases are enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral-asparaginase in the presence of L-Asp was determined at 2.5 A resolution and refined to an R cryst of 19.2 (R free = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well.
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PMID:Structural and functional insights into Erwinia carotovora L-asparaginase. 1864 44

The L-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leads to L-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards L-asparagine are highly desirable as better alternatives in cancer therapy. L-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16-19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 A resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 A and one molecule of L-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure-activity relationship in L-asparaginases.
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PMID:Expression, purification and crystallization of Helicobacter pylori L-asparaginase. 1867 46

Bacterial L-asparaginases are amidohydrolases that catalyse the conversion of L-asparagine to L-aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel L-asparaginase from the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for L-asparagine over L-glutamine and, in contrast to most L-asparaginases, it exhibited a sigmoidal behaviour towards L-glutamine. The enzyme preserved full activity after 2 h incubation at 45 degrees C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme, AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications.
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PMID:Helicobacter pyloril-asparaginase: a promising chemotherapeutic agent. 1898 25

Asparaginase from Helicobacterpylori has been cloned and expressed in E. coli cells. Optimization of culturing and expression conditions allowed achieving stable synthesis of catalytically active asparaginase amounting up to 6% of total bacterial protein. A method developed for enzyme purification included a single chromatographic stage and provided more than sixty percent yield of homogeneous asparaginase. Specific asparaginase and glutaminase activities were estimated to 92 and 8,3 x 10(-3) ME/mg respectively. Due to low glutaminase specificity HpA may be employed as a non-toxic drug for leukemia treatment.
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PMID:[Cloning, expression and purification of Helicobater pylori L-asparaginase]. 1898 65


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