Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Distasio, John A.; Niederman, Robert A.; Kafkewitz, David; Goodman, David B. P.

doi:10.1016/s0021-9258(17)32924-1

Cited by 80 publications

(11 citation statements)

References 25 publications

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“…Numerous studies of L-asparaginases have been conducted in order to understand the catalytic mechanism and the substrate specificity of these enzymes. Studies of the dependence of activity on pH, conducted for various Lasparaginases, confirmed that the enzymes are stable and active in the pH range of 4-9 (25)(26)(27). It has been shown that the enzymatic reaction proceeds according to a twostep ping-pong mechanism similar to the mechanism of serine proteases (28), except that the attacking nucleophile is a threonine.…”

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confidence: 88%

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,

2001

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Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity.

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confidence: 88%

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,

2001

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“…Substrate Selectivity of Recombinant Asparaginase. l -Asparaginases typically demonstrate some catalytic activity for d -asparagine and l -glutamine in addition to their preferred substrate, l -asparagine. − The selectivity of recombinant asparaginase from A. fulgidus for d - and l -asparagine was evaluated at 37 and 70 °C using 0.2 unit of the enzyme. It was found that the enzyme has 5-fold higher activity for l - than for d -asparagine.…”

Section: Resultsmentioning

confidence: 99%

Biosensor for Asparagine Using a Thermostable Recombinant Asparaginase from Archaeoglobus fulgidus

Wang

Bachas

2002

Anal. Chem.

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Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 degrees C. The KM for L-asparagine was 8 x 10(-5) and 5 x 10(-6) M at 37 and 70 degrees C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 x 10(-5) M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability.

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“…Wolinella succinogenes derived L-ASNase ( WoA ) was initially reported as a glutaminase-free L-ASNase variant. Reinert et al compared this variant with E. coli derived L-ASNase and showed that WoA did not suppress the immune response in mice and lacked hepatotoxicity [ 159 , 160 , 161 , 162 ]. In addition, it was demonstrated that PEG- WoA caused less toxic side effects compared to PEGylated E. coli L-ASNase, suggesting that WoA would potentially be a safer drug due to its glutaminase-free properties [ 163 ].…”

Section: The Development Of Novel L-asnase Variantsmentioning

confidence: 99%

Novel Insights on the Use of L-Asparaginase as an Efficient and Safe Anti-Cancer Therapy

Trimpont

Peeters

Visser

et al. 2022

Cancers

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L-Asparaginase (L-ASNase) is an enzyme that hydrolyses the amino acid asparagine into aspartic acid and ammonia. Systemic administration of bacterial L-ASNase is successfully used to lower the bioavailability of this non-essential amino acid and to eradicate rapidly proliferating cancer cells with a high demand for exogenous asparagine. Currently, it is a cornerstone drug in the treatment of the most common pediatric cancer, acute lymphoblastic leukemia (ALL). Since these lymphoblasts lack the expression of asparagine synthetase (ASNS), these cells depend on the uptake of extracellular asparagine for survival. Interestingly, recent reports have illustrated that L-ASNase may also have clinical potential for the treatment of other aggressive subtypes of hematological or solid cancers. However, immunogenic and other severe adverse side effects limit optimal clinical use and often lead to treatment discontinuation. The design of optimized and novel L-ASNase formulations provides opportunities to overcome these limitations. In addition, identification of multiple L-ASNase resistance mechanisms, including ASNS promoter reactivation and desensitization, has fueled research into promising novel drug combinations to overcome chemoresistance. In this review, we discuss recent insights into L-ASNase adverse effects, resistance both in hematological and solid tumors, and how novel L-ASNase variants and drug combinations can expand its clinical applicability.

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Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Cited by 80 publications

References 25 publications

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,

Biosensor for Asparagine Using a Thermostable Recombinant Asparaginase from Archaeoglobus fulgidus

Novel Insights on the Use of L-Asparaginase as an Efficient and Safe Anti-Cancer Therapy

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Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Cited by 80 publications

References 25 publications

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase,

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase,

Biosensor for Asparagine Using a Thermostable Recombinant Asparaginase from Archaeoglobus fulgidus

Novel Insights on the Use of L-Asparaginase as an Efficient and Safe Anti-Cancer Therapy

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Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,

Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,