Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy.

Swain, Amy L.; Jaskólski, Mariusz; Housset, D.; Rao, Jayasimha; Wlodawer, Alexander

doi:10.1073/pnas.90.4.1474

Cited by 267 publications

(348 citation statements)

References 26 publications

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“…Model rebuilding was performed using the FRODO computer graphics package [24]. The structure was solved by molecular replacement using the EcA tetramer [10] as a model. Rotation and translation functions gave very clear solutions for different sets of parameters.…”

Section: Methodsmentioning

confidence: 99%

“…The crystal structures of five bacterial asparaginases have been solved (EcA [10], ErA [11], AGA [12], Pseudomonas 7A glutaminase-asparaginase (PGA) [13], and Wolinella succinogenes asparaginase (WsA) (Lubkowski et al, submitted for publication). Each has the same tetrameric quaternary structure as it has in solution, a homotetramer of approx.…”

Section: ! Introductionmentioning

confidence: 99%

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A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Palm¹,

Łubkowski²,

Derst³

et al. 1996

FEBS Letters

112

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Escherichia coli asparaginase |I catalyzes the hydrol-) sis of L-asparagine to L-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.:£ey words'." Asparaginase II; Acyl-enzyme intermediate; !'hreonine amidohydrolase; Enzymatic mechanism !. IntroductionAsparaginases catalyze the hydrolysis of L-asparagine to L~spartate and ammonia. The enzymes isolated from Escheri-'hia coli (EcA) and Erwinia chrysanthemi (ERA) have been ltilized as anti-leukemia drugs for many years [1]. Treatment vith asparaginases is often accompanied by severe side effects, vhich are partially attributed to the glutaminase activity of hese enzymes [2]. In order to understand the enzyme specifi-:ity and to ultimately eliminate the glutaminase activity, the nechanism of action must be elucidated. Several members of t larger family of homologous L-asparaginases have thus been horoughly investigated over many years.Results of kinetic measurements [3,4] indicated that the en-,ymatic reaction proceeds via a covalent intermediate, probtbly a ~-aspartyl enzyme (Fig. 1). This mechanism was con-,irmed by NMR studies with aspartate through the oxygen ~xchange reaction with bulk solvent at the side chain carboxdate [5]. These experiments showed that at pH < 5 L-aspartate ~ith a protonated side chain binds to the enzyme as tightly as ~-asparagine and it can also form an acyl-enzyme intermediate, subsequently hydrolyzed to L-aspartate. Thus, at low pH 3oth L-aspartate and L-asparagine can function as substrates. l'he nature and identity of the primary nucleophile of the mzyme participating in the formation of the acyl-enzyme in-*Corresponding author. Fax: (1) . Each has the same tetrameric quaternary structure as it has in solution, a homotetramer of approx. 4x330 residues. The tetramer is more accurately described as a dimer of dimers. Each of two identical active sites in each dimer is formed by both monomers. In the structure the active sites have been observed with and without aspartate as ligand. Part of the active site is covered by a flexible loop that contains the two important residues Thr-12 and Tyr-25. The hydroxyl groups of Thr-12 and Thr-89 are closest to the side chain carboxylate of an aspartate bound in the active site. These residues are the most likely candidates for the primary nucleophile. Bacterial L-asparaginases were the first threonine amidohydrolases described in the literature. Only recently, two other threonine amidohydrolases, 20S proteasome [14] and aspartylglucosaminidase [15], have been reported. Both enzymes belong t...

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Section: Methodsmentioning

confidence: 99%

Section: ! Introductionmentioning

confidence: 99%

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Palm¹,

Łubkowski²,

Derst³

et al. 1996

FEBS Letters

112

View full text Add to dashboard Cite

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“…P06608) [29] and E. coli (EcA2; NCBI no. 1NNSA) [3] The residues conserved in all three L-asparaginases or in either of two enzymes are shown in black and grey boxes respectively. The fundamental residues involved in substrate binding are marked with arrows and the position of the nucleophile is indicated by an asterisk.…”

Section: Resultsmentioning

confidence: 99%

“…These enzymes also hydrolyse L-Gln, but with a lower rate compared with L-Asn. The active-site residues have been identified in the crystallographic studies of asparaginase from Eschericha coli [3]. The high-resolution structure of 0.17 nm (1.7 Å ) for L-asparaginase from Erwinia chrysanthemi elucidated the residues presumably responsible for different specificities towards two physiological substrates, L-Asn and L-Gln [4].…”

Section: Introductionmentioning

confidence: 99%

One‐step purification and kinetic properties of the recombinant l‐asparaginase from Erwinia carotovora

Krasotkina

Borisova

Gervaziev

et al. 2004

Biotech and App Biochem

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ECAR-LANS, the recombinant L-asparaginase fromErwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS. More than 90 % purity, complemented with 72 % active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified L-asparaginase was 630 i.u./mg. The ECAR-LANS K m value was 98 × 10 −6 M for the main physiological substrate L-Asn and 3400 × 10 −6 M for L-Gln. ECAR-LANS was found to have low relative glutaminase activity (1.2 %) at physiological concentrations of L-Asn and L-Gln in blood. Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.

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“…Asparginase (EC 3.5.1.1) is known to be one of the most important bed rocks of ALL treatment in almost all pediatric regimens of youth and adults' treatment protocols (4). Various prokaryotic and eukaryotic resources that are able to provide this enzyme include different kinds of Bacteria, Algae, Fungi (4) and Yeasts play an important role in amino acids metabolism, and utilization and maintaining nitrogen balance within cells.…”

Section: Introductionmentioning

confidence: 99%

Applying Bioinformatic Tools for Modeling and Modifying Type II E. coli l-Asparginase to Present a Better Therapeutic Agent/Drug for Acute Lymphoblastic Leukemia

et al. 2017

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Background: Asparginase is known to be one of the most important bedrocks of acute lymphoblastic leukemia (ALL) treatment in almost all pediatric regimens in treatment protocols. Escherichia coli L-Asparginase (EC 3.5.1.1) is one of the most common resources to produce this enzyme. One of the affordable methods to overcome the side effects of drug is utilizing bioinformatic tools in the form of In silico study. In this study we designed a new structure of L-Asparginase to decrease its toxicity, reduce some side effects and increase the stability. Methods: We used some bioinformatics software and servers like Toxin red, Popmusic, kobami and I-TASSER server to reduce toxicity level of enzyme, and to increase stability and enzyme half-life. Results: We obtained 6 protein sequences in which the best was Mut 6 with four changes in structure: L23G, K129L, S263C and R291F. In contrast to the wild type, the new predicted protein is not toxic and has 25 hours more half-life and 600 kcal/mol more stable with no significant change in protein secondary, tertiary structure, antigenicity and allergenicity. Conclusions: Finally, sequence number 6 was the only sequence with all distinct characteristics: non-toxic, more stability and more half life.

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Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy.

Cited by 267 publications

References 26 publications

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

One‐step purification and kinetic properties of the recombinant l‐asparaginase from Erwinia carotovora

Applying Bioinformatic Tools for Modeling and Modifying Type II E. coli l-Asparginase to Present a Better Therapeutic Agent/Drug for Acute Lymphoblastic Leukemia

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