Overexpression and Purification of Asparagine Synthetase from<i>Escherichia coli</i>

Sugiyama, Akio; Kato, Hiroaki; Nishi­oka, Takaaki; Oda, Jun’ichi

doi:10.1271/bbb.56.376

Cited by 15 publications

(22 citation statements)

References 5 publications

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“…On this point, we note that there is no evolutionary relationship of glutamine-and ammonia-dependent asparagine synthetases, the latter family being related to aminoacyl tRNA synthetases (172,173). Incubation of 11 with AS-A, the ammonia-dependent ASNS isolated from Escherichia coli (174,175), however, showed that this compound was a poor (millimolar) inhibitor because it was not adenylated when bound within the synthetase active site. A synthetic route to the required adenylated sulfoximine was therefore developed by Hiratake and coworkers, yielding the target compound as a mixture of diastereoisomers (7a and 7b) (Figure 6) at the sulfur center (169).…”

Section: A Nanomolar Inhibitor Of Human Asparagine Synthetasementioning

confidence: 91%

Asparagine Synthetase Chemotherapy

Richards

Kilberg

2006

Annu. Rev. Biochem.

204

221

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Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzymebased methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. Significant side effects can arise in these protocols and, in many cases, patients develop drug-resistant forms of the disease that may be correlated with up-regulation of the enzyme glutamine-dependent asparagine synthetase (ASNS). Though the precise molecular mechanisms that result in the appearance of drug resistance are the subject of active study, potent ASNS inhibitors may have clinical utility in treating asparaginase-resistant forms of childhood ALL. This review provides an overview of recent developments in our understanding of (a) the structure and catalytic mechanism of ASNS, and (b) the role that ASNS may play in the onset of drug-resistant childhood ALL. In addition, the first successful, mechanism-based efforts to prepare and characterize nanomolar ASNS inhibitors are discussed, together with the implications of these studies for future efforts to develop useful drugs.

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Section: A Nanomolar Inhibitor Of Human Asparagine Synthetasementioning

confidence: 91%

Asparagine Synthetase Chemotherapy

Richards

Kilberg

2006

Annu. Rev. Biochem.

204

221

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“…Asparagine synthetase (L-aspartate:ammonia ligase (AMPforming), EC 6.3.1.1) is an enzyme that catalyzes the synthesis of asparagine from aspartate using adenosine triphosphate (ATP) as the energy source in the presence of a nitrogen donor (4). The nitrogen donor can be glutamine or ammonia.…”

mentioning

confidence: 99%

Identification and Functional Characterization of a Novel Bacterial Type Asparagine Synthetase A

Manhas¹,

Tripathi²,

Khan³

et al. 2014

Journal of Biological Chemistry

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Background: Asparagine synthetase A catalyzes the synthesis of asparagine from aspartate in the presence of ammonia as a nitrogen donor. Results: L. donovani ASNA enzyme is of bacterial origin and is both ammonia-and glutamine-dependent. Conclusion: LdASNA is essential for survival of the Leishmania parasite. Significance: The absence of LdASNA homologs from humans validates Leishmania ASNA as a novel drug target.

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“…The resulting DNA sequence was checked by dideoxy sequencing (Sanger, Nicklen & Coulson, 1977). The mutant gene was subcloned into the expression vector, pUNAd37 (Sugiyama et al, 1992).…”

Section: Mutagenesismentioning

confidence: 99%

“…The overexpression in E. coli and the purification were carried out by the method described previously (Sugiyama et al, 1992), followed by hydrophobic interaction chromatography on a PhenyI-TOYOPEARL 650 column. The last step of purification was as follows.…”

Section: Overexpression and Purificationmentioning

confidence: 99%

“…The asnA gene from E. coli coded for a polypeptide of 330 amino-acid residues with a molecular weight of 36 700, and the asnA enzyme exists as a dimer of identical subunits (Nakamura et al, 1981;Sugiyama, Kato, Nishioka & Oda, 1992). Each subunit contains two Cys residues, Cys51 and Cys315, and their chemical modification causes inactivation of the enzymatic activity (Cedar & Schwartz, 1969a).…”

Section: Introductionmentioning

confidence: 99%

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Crystallization and preliminary crystallographic study of asparagine synthetase fromEscherichia coli

Nakatsu

Kato²,

Oda³

1996

Acta Cryst D

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Crystals of Escherichia coli asparagine synthetase have been obtained from 45% saturated ammonium sulfate solution at pH 7.5 by using its Cys-free (Cys51 ---,Ala, Cys315~Ala) mutant. The crystals belong to space group P21, with cell dimensions a = 52.90(4), b = 126.2 (2), c = 52.8 (1),~, and fl = 105.3(1) °. The self-rotation function specifies that there is one dimer in the asymmetric unit and the subunits are related by a noncrystallographic twofold axis. Complete data sets up to 2.7 A, resolution have been collected on an R-AXIS IIc imaging-plate system.

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Overexpression and Purification of Asparagine Synthetase fromEscherichia coli

Cited by 15 publications

References 5 publications

Asparagine Synthetase Chemotherapy

Asparagine Synthetase Chemotherapy

Identification and Functional Characterization of a Novel Bacterial Type Asparagine Synthetase A

Crystallization and preliminary crystallographic study of asparagine synthetase fromEscherichia coli

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