Expression, purification and crystallization of<i>Helicobacter pylori</i><scp>L</scp>-asparaginase

Dhavala, Prathusha; Krasotkina, Julya; Dubreuil, Christine; Papageorgiou, Anastassios C.

doi:10.1107/s1744309108020186

Cited by 13 publications

(6 citation statements)

References 21 publications

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“…Recent reports have shown that Erwinia carotovora L-asparaginase (EwA) and two Helicobacter pylori LASNases have reduced (1.5%) [46] to negligible (0.01%) [47] glutaminase activities, respectively. Sequence comparison indicates that the active site is highly conserved between L-ASNases with only a few variations.…”

Section: Structural Basis Of Asparaginase-glutaminase Activity Ratiomentioning

confidence: 99%

Structure-Function Relationships and Clinical Applications of L-Asparaginases

Labrou¹,

Papageorgiou²,

Avramis³

2010

CMC

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L-asparaginase (L-ASNase, EC 3.5.1.1) catalyzes the hydrolysis of the non-essential amino acid L-Asn to LAsp and ammonia and is widely used for the treatment of haematopoetic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas. Therapeutic forms of L-ASNase come from different biological sources (primarily E. coli and Erwinia chrysanthemi). It is well established that the various preparations have different biochemical pharmacology properties, and different tendency to induce side-effects. This is due to different structural, physicochemical and kinetic properties of L-ASNases from the various biological sources. Understanding these properties of various L-ASNases would allow a better decipherment of their catalytic and therapeutic features, thus enabling more accurate predictions of the behaviour of these enzymes under a variety of therapeutic conditions. In addition, detailed understanding of the catalytic mechanism of L-ASNases might permit the design of new forms of L-ASNases with optimal biochemical properties for clinical applications. In this paper we review the available biochemical and pharmacokinetic information of the therapeutic forms of bacterial L-ASNases, and focus on a detailed description of structure, function and clinical applications of these enzymes.

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Section: Structural Basis Of Asparaginase-glutaminase Activity Ratiomentioning

confidence: 99%

Structure-Function Relationships and Clinical Applications of L-Asparaginases

Labrou¹,

Papageorgiou²,

Avramis³

2010

CMC

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“…In addition to the biochemically well-characterized enzymes described above, PDB currently holds two structures of type II ASNase from H. pylori (HpA; PDB IDs 2wt4 and 2wlt) [59,108] and one structure of C. jejuni ASNase (CjA, PDB ID 3nxk, unpublished) (Table 4). Unfortunately, in neither case structural reports were complemented by activity/kinetic characteristics, limiting subsequent analysis.…”

Section: Pseudomonas 7a (Pga)mentioning

confidence: 99%

Structural and biochemical properties of L‐asparaginase

Łubkowski

Wlodawer

2021

The FEBS Journal

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L-asparaginase (a hydrolase converting L-asparagine to L-aspartic acid) was the first enzyme to be used in clinical practice as an anticancer agent after its approval in 1978 as a component of a treatment protocol for childhood acute lymphoblastic leukemia (ALL). Structural and biochemical properties of L-asparaginases have been extensively investigated during the last half century, providing an accurate structural description of the enzyme isolated from a variety of sources, as well as clarifying the mechanism of its activity. This review provides a critical assessment of the current state of knowledge of primarily structural, but also selected biochemical properties of "bacterial-type" L-asparaginases from different organisms. The most extensively studied members of this enzyme family are L-asparaginases highly homologous to one of the two enzymes from Escherichia coli (usually referred to as EcAI and EcAII). Members of this enzyme family, although often called bacterial-type L-asparaginases, have been also identified in such divergent organisms as archaea or eukarya. Over 100 structural models of L-asparaginases have been deposited in the Protein Data Bank during the last 30 years. One of the prime achievements of structure-centered approaches was the elucidation of the details of the mechanism of enzymatic action of this unique hydrolase that utilizes a side chain of threonine as the primary nucleophile. The molecular basis of other important properties of these enzymes, such as their substrate specificity, are still being evaluated. Results of structural and mechanistic studies of L-asparaginases are being utilized in efforts to improve the clinical properties of this important anticancer drug.

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“…HpA was expressed and purified using ion-exchange chromatography as reported previously (Dhavala et al, 2008). Crystals were grown using the hanging-drop vapour-diffusion method at 289 K by mixing 2.5 ml enzyme solution (3.7 mg ml À1 in 10 mM HEPES-NaOH pH 7.0) with an equal volume of a reservoir solution consisting of 17.5%(w/v) PEG 4000, 0.1 M magnesium formate, 0.1 M HEPES-NaOH pH 7.0.…”

Section: Crystallization Conditionsmentioning

confidence: 99%

Structure ofHelicobacter pyloriL-asparaginase at 1.4 Å resolution

Dhavala

Papageorgiou

2009

Acta Crystallogr D Biol Cryst

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Bacterial L-asparaginases have been used in the treatment of childhood acute lymphoblastic leukaemia for over 30 years. Their therapeutic effect is based on their ability to catalyze the conversion of L-asparagine, an essential amino acid in certain tumours, to L-aspartic acid and ammonia. Two L-asparaginases, one from Escherichia coli and the other from Erwinia chrysanthemi, have been widely employed in clinical practice as anti-leukaemia drugs. However, L-asparaginases are also able to cause severe side effects owing to their intrinsic glutaminase activity. Helicobacter pylori L-asparaginase (HpA) has been reported to have negligible glutaminase activity. To gain insight into the properties of HpA, its crystal structure in the presence of L-aspartate was determined to 1.4 A resolution, which is one of the highest resolutions obtained for an L-asparaginase structure. The final structure has an R(cryst) of 12.6% (R(free) = 16.9%) with good stereochemistry. A detailed analysis of the active site showed major differences in the active-site flexible loop and in the 286-297 loop from the second subunit, which is involved in active-site formation. Accordingly, Glu289, Asn255 and Gln63 are suggested to play roles in modulating the accessibility of the active site. Overall, the structural comparison revealed that HpA has greater structural similarity to E. coli L-asparaginase than to any other L-asparaginase, including Er. carotovora L-asparaginase, despite the fact that the latter is also characterized by low glutaminase activity.

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Expression, purification and crystallization ofHelicobacter pyloriL-asparaginase

Cited by 13 publications

References 21 publications

Structure-Function Relationships and Clinical Applications of L-Asparaginases

Structure-Function Relationships and Clinical Applications of L-Asparaginases

Structural and biochemical properties of L‐asparaginase

Structure ofHelicobacter pyloriL-asparaginase at 1.4 Å resolution

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