Crystallization and preliminary crystallographic analysis ofL-asparaginase fromErwinia carotovora

Abstract

Bacterial l-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia for over 30 y. However, their use is limited owing to the glutaminase activity of the administered enzymes, which results in serious side effects. In contrast, l-asparaginase from Erwinia carotovora exhibits low glutaminase activity at physiological concentrations of l-asparagine and l-glutamine in the blood. Recombinant Er. carotovora l-asparaginase was crystallized in the presence of l-glutamate by the hangingdrop vapour-diffusion method using 10 mg ml À1 purified enzyme, 16-18%(w/v) PEG 3350 and 0.2 M NaF. X-ray diffraction data were collected to 2.6 Å at 293 K using an in-house rotating-anode generator. The crystals belong to the monoclinic P2 1 space group, with unit-cell parameters a = 78.0, b = 112.3, c = 78.7 Å , = 101.9and a homotetramer in the crystallographic asymmetric unit. A molecular-replacement solution has been found and refinement is currently in progress. The crystal structure may provide leads towards proteinengineering efforts aimed at safer asparaginase administration in leukaemia treatment.

Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells

“…Asparaginase is a well known chemotherapeutic enzyme, universally used in the treatment protocols of various neoplasms, particularly of lymphoid origins [ 30 ]. The commercially available asparaginases possesses 3–10% intrinsic glutaminase activity [ 20 ], which often produces several side effects including pancreatitis, hyperglycemia, neurological seizures and various hypersensitivity reactions [ 31 ]. Therefore, the search of glutaminase free asparaginase with effective antineoplastic activity has gained significant interest of modern researchers.…”

Tailoring structure–function properties of L-asparaginase: engineering resistance to trypsin cleavage

“…These seven residues are probably the targets of chemical modification by mPEG-SNHS. Analysis of the crystal structure of the enzyme showed that none of these lysine residues is located in regions of the protein implicated in substrate binding and catalysis, with the exception of Lys 30 , which lies at the flexible loop (residues 10-40, Figure 1B) near to the enzyme active site [29,31,35]. Site-directed mutagenesis of Lys 30 to alanine gave a mutant enzyme with kinetic parameters very close to the wild-type enzyme (K m = 0.075 + − 0.03 mM for Lasparagine).…”

“…All asparaginases are homotetramers with 222-symmetry and a molecular mass in the range 140-150 kDa, with a highly conserved overall fold [29][30][31]. They are composed of four identical subunits denoted A, B, C and D [32].…”

L‐asparaginase isolated fromStreptomyces ansochromogenespromotes Th1 profile and activatesCD8+T cells in humanPBMC: anin vitroinvestigation

“…; Müller and Boos ; Wikman et al . ) and immunosuppression reactions (Pinheiro and Boos ; Avramis and Tiwari ).…”