Expression, purification and crystallization of Helicobacter pylori L-asparaginase.

The L-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leads to L-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards L-asparagine are highly desirable as better alternatives in cancer therapy. L-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16-19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 A resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 A and one molecule of L-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure-activity relationship in L-asparaginases.

Structural and functional insights into Erwinia carotovora L-asparaginase.

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 A resolution and refined to an R cryst of 19.2 (R free = 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.

Crystallization and preliminary crystallographic analysis of L-asparaginase from Erwinia carotovora.

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 hanging-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 16-18%(w/v) PEG 3350 and 0.2 M NaF. X-ray diffraction data were collected to 2.6 A 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 A, beta = 101.9 degrees and 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 protein-engineering efforts aimed at safer asparaginase administration in leukaemia treatment.

One-step purification and kinetic properties of the recombinant L-asparaginase from Erwinia carotovora.

ECAR-LANS, the recombinant L-asparaginase from Erwinia 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 98x10(-6) M for the main physiological substrate L-Asn and 3400x10(-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.

Hemophilia A and B are associated with abnormal spatial dynamics of clot growth.

To gain greater insight into the nature of the bleeding tendency in hemophilia, we compared the spatial dynamics of clotting in platelet-free plasma from healthy donors and from patients with severe hemophilia A or B (factor VIII:C or IX:C<1%). Clotting was initiated via the intrinsic or extrinsic pathway in a thin layer of nonstirred plasma by bringing it in contact with the glass or fibroblast monolayer surface. The results suggest that clot growth is a process consisting of two distinct phases, initiation and elongation. The clotting events on the activator surface and the preceding period free of visible signs of clotting are the initiation phase. In experiments with and without stirring alike, this phase is prolonged in hemophilic plasma activated by the intrinsic, but not the extrinsic pathway. Strikingly, both hemophilia A and B are associated with a significant deterioration in the elongation phase (clot thickening), irrespective of the activation pathway. The rate of clot growth in hemophilic plasma is significantly lower than normal and declines quickly. The resulting clots are thin, which may account for the bleeding disorder.