Structural comparison of<i>Escherichia coli</i><scp>L</scp>-asparaginase in two monoclinic space groups

Sanches, Mário; Barbosa, J.A.R.G.; Oliveira, R.T. de; Neto, José Abrahão; Polikarpov, Igor

doi:10.1107/s0907444902021200

Cited by 43 publications

(44 citation statements)

References 21 publications

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“…The main chain RMSD of Cα atoms between the structure solved in our laboratory (PDB code: https://doi.org/10.2210/pdb6EOK/pdb) and three (1NNS, 3ECA and 1JAZ) of the several structures, available in the protein data bank, is generally low (Figure S5). The above‐mentioned region, not accounted by any electron density, corresponds to a long loop (the so called “lid”) which closes the catalytic site and clearly experiences a large flexibility in solution . It is interesting to point out that our protein and 1JAZ have been crystallized in the absence of the substrate/product (asparagine/aspartate), while the structures 1NNS and 3ECA have been obtained from ANSII co‐crystallized with aspartate.…”

Section: Resultsmentioning

confidence: 99%

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

Cerofolini

Giuntini

Carlon

et al. 2019

Chemistry A European J

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Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to “evade” the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X‐ray crystallography.

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Section: Resultsmentioning

confidence: 99%

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

Cerofolini

Giuntini

Carlon

et al. 2019

Chemistry A European J

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“…Other reports on production and purification of L-asparaginase from E. coli revealed that the molecular weight was determined as 153 KDa with the help of SDS-PAGE43. The functional L-asparaginase from E. coli is a homotetramer with a molecular weight of about 142 kDa44. Purified L-asparaginase from Streptomyces gulbargensis 11, Streptomyces albidoflavus 9, Streptomyces PDK223 and Streptomyces noursei 15 exhibited a molecular weight of 85, 112, 140 kDa and 102 kDa, respectively.…”

Section: Resultsmentioning

confidence: 99%

Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82

El‐Naggar

Deraz

Soliman

et al. 2016

Sci Rep

108

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L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml−1min−1, respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS–PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.

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“…The molecular structure of the E coli asparaginase active site in complex with aspartic acid was first revealed in the 3ECA x-ray crystal structure 31 and later in a higher resolution structure, 1NNS. 32 Those structures revealed contacts between S58, Q59, D90, E283, and backbone groups of aspartic acid. N246 and N248 did not contact either substrate directly, despite the possibility that N248 might stabilize E283 in proximity of the ligand.…”

Section: Discussionmentioning

confidence: 99%

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Chan

Lorenzi

Anishkin

et al. 2014

Blood

150

131

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• We used molecular dynamics, saturation mutagenesis, and enzymologic screening to develop a glutaminase-free mutant (Q59L) L-ASP.• We then used Q59L to show that glutaminase activity is not required for L-ASP activity against ASNS-negative cancer cells.L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types.Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers. (Blood. 2014;123(23):3596-3606) Introduction L-Asparaginase (L-ASP) is an enzyme drug used in combination with vincristine and a glucocorticoid (eg, dexamethasone) to treat acute lymphoblastic leukemia (ALL).1,2 We 3-6 and others 7 have reported a rationale for testing L-ASP against low-asparagine synthetase (ASNS) solid tumors as well. L-ASP's primary known enzymatic activity is deamidation of asparagine to aspartic acid and ammonia, but it also deamidates glutamine to glutamic acid and ammonia, although with lower affinity and lower maximal rate. L-ASP therapy is often limited by toxic side effects that are generally attributed to the glutaminase activity. 8,9 Those side effects often preclude completion of the full treatment regimen, resulting in poor outcome. 10 The question that arises, however, is whether the therapeutic index of L-ASP could be increased by decreasing its glutaminase activity 8,9 or whether that would also decrease the anticancer effect commensurately.One side of the debate hypothesizes that L-ASP's therapeutic index can be improved by increasing glutaminase activity. In support of that hypothesis, data collected over the last decade have suggested that glutaminase activity generally increases the efficacy of L-ASP and is sometimes required to achieve an anticancer effect. Those studies have reported asparaginase activity to be expendable. [11][12][13][...

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Structural comparison ofEscherichia coliL-asparaginase in two monoclinic space groups

Cited by 43 publications

References 21 publications

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

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