Structural and functional insights into Erwinia carotovora l‐asparaginase

Abstract

Bacterial l‐asparaginases are enzymes that catalyze the hydrolysis of l‐asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low‐rate glutaminase activity, however, causes serious side‐effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side‐effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral‐asparaginase in the presence of l‐Asp was determined at 2.5 Å resolution and refined to an Rcryst of 19.2 (Rfree = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well.

Structure-Function Relationships and Clinical Applications of L-Asparaginases

“…This model, however, proved useful in molecular replacement trials that led to the crystal structure determination of EcAII at 2.3 Å resolution in 1993 [34]. Subsequently, crystal structures of a number of LASNases were determined, including ErA [35], Wolinella succinogenes L-ASNase (WsA) [36], Erwinia carotovora (EwA) [37,38] and related asparaginase-glutaminases from Acinetobacter glutaminasificans (AgA) [23] and Pseudomonas fluorescence (PgA) [22] as shown in Fig. (3).…”

Antitumor activity of L-asparaginase from Erwinia Carotovora from against different leukemic and solid tumours cell lines

“…При культивировании клеток в среде с низким содержанием сыворотки (0,1% ЭТС) апоптоз в этих клетках обнаруживается уже через 8 ч культивирования [18]. При предварительной синхронизации клеток перед добавлением L-аспарагиназы также резко увеличивается гибель клеток в результате апоптоза [19], скорее всего потому, что в этих случаях в опухолевых клетках не может осуществляться синтез антиапоптотических белков Bcl-2. Способность клеток синтезировать белок в наших экспериментах при их культивировании в присутствии L-аспарагиназы в полной ростовой среде, содержащей 10% ЭТС, может быть связана c тем, что здесь происходит извлечение аминокислот из белков ЭТС при их ферментативном гидролизе в ростовой среде ферментами, поступившими из лизосом погибших клеток.…”

Isolation and Purification of High Efficiency L-asparaginase by Quantitative Preparative Continuous-elution SDS PAGE Electrophoresis

“…In addition, inspiration of the establishment with reducing agents like reduced glutathione (GSH), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) and inhibition in the presence of thiol group blocking reagents, specifically, P-chloro mercury benzoate and iodiacetate provided supplementary confirmation for the role of sulfhydryl groups in the catalytic activity of the enzyme. The enzyme entirely lost its activity at 5.0M urea [35] and only 5.0M sodium dodecyl sulfate (SDS).…”