Project description:Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects.
Project description:l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ?10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemi l-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations-open and closed-corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.
Project description: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.
Project description:A Bacillus licheniformis isolate with high L-asparaginase productivity was recovered upon screening two hundred soil samples. This isolate produces the two types of bacterial L-asparaginases, the intracellular type I and the extracellular type II. The catalytic activity of type II enzyme was much higher than that of type I and reached about 5.5 IU/ml/h. Bioinformatics analysis revealed that L-asparaginases of Bacillus licheniformis is clustered with those of Bacillus subtilis, Bacillus haloterans, Bacillus mojavensis and Bacillus tequilensis while it exhibits distant relatedness to L-asparaginases of other Bacillus subtilis species as well as to those of Bacillus amyloliquefaciens and Bacillus velezensis species. Upon comparison of Bacillus licheniformis L-asparaginase to those of the two FDA approved L-asparaginases of E. coli (marketed as Elspar) and Erwinia chrysanthemi (marketed as Erwinaze), it observed in a cluster distinct from- and with validly predicted antigenic regions number comparable to those of the two mentioned reference strains. It exhibited maximum activity at 40 °C, pH 8.6, 40 mM asparagine, 10 mM zinc sulphate and could withstand 500 mM NaCl and retain 70% of its activity at 70 °C for 30 min exposure time. Isolate enzyme productivity was improved by gamma irradiation and optimized by RSM experimental design (Box-Behnken central composite design). The optimum conditions for maximum L-asparaginase production by the improved mutant were 39.57 °C, 7.39 pH, 20.74 h, 196.40 rpm, 0.5% glucose, 0.1% ammonium chloride, and 10 mM magnesium sulphate. Taken together, Bacillus licheniformis L-asparaginase can be considered as a promising candidate for clinical application as antileukemic agent.
Project description:L-Asparaginase (L-asparagine aminohydrolase, E.C. 188.8.131.52) has been proven to be competent in treating Acute Lymphoblastic Leukaemia (ALL), which is widely observed in paediatric and adult groups. Currently, clinical L-Asparaginase formulations are derived from bacterial sources such as Escherichia coli and Erwinia chrysanthemi. These formulations when administered to ALL patients lead to several immunological and hypersensitive reactions. Hence, additional purification steps are required to remove toxicity induced by the amalgamation of other enzymes like glutaminase and urease. Production of L-Asparaginase that is free of glutaminase and urease is a major area of research. In this paper, we report the screening and isolation of fungal species collected from the soil and mosses in the Schirmacher Hills, Dronning Maud Land, Antarctica, that produce L-Asparaginase free of glutaminase and urease. A total of 55 isolates were obtained from 33 environmental samples that were tested by conventional plate techniques using Phenol red and Bromothymol blue as indicators. Among the isolated fungi, 30 isolates showed L-Asparaginase free of glutaminase and urease. The L-Asparaginase producing strain Trichosporon asahii IBBLA1, which showed the highest zone index, was then optimized with a Taguchi design. Optimum enzyme activity of 20.57 U mL-1 was obtained at a temperature of 30?°C and pH of 7.0 after 60?hours. Our work suggests that isolation of fungi from extreme environments such as Antarctica may lead to an important advancement in therapeutic applications with fewer side effects.
Project description:An exhaustive screening program was applied for scoring a promising L-asparaginase producing-isolate. The recovered isolate was identified biochemically and molecularly and its L-asparaginase productivity was optimized experimentally and by Response Surface Methodology. The produced enzyme was characterized experimentally for its catalytic properties and by bioinformatics analysis for its immunogenicity. The promising L-asparaginase producing-isolate was selected from 722 recovered isolates and identified as Stenotrophomonas maltophilia and deposited at Microbiological Resources Centre (Cairo Mircen) under the code EMCC2297. This isolate produces both intracellular (type I) and extracellular (type II) L-asparaginases with about 4.7 fold higher extracellular L-asparaginase productivity. Bioinformatics analysis revealed clustering of Stenotrophomonas maltophiliaL-asparaginase with those of Pseudomonas species and considerable closeness to the two commercially available L-asparaginases of E. coli and Erwinia chrysanthemi. Fourteen antigenic regions are predicted for Stenotrophomonas maltophiliaL-asparaginase versus 16 and 18 antigenic regions for the Erwinia chrysanthemi and E. coliL-asparaginases. Type II L-asparaginase productivity of the test isolate reached 4.7 IU/ml/h and exhibited maximum activity with no metal ion requirement at 37 °C, pH 8.6, 40 mM asparagine concentration and could tolerate NaCl concentration up to 500 mM and retain residual activity of 55% at 70 °C after half an hour treatment period. Application both of random mutation by gamma irradiation and Response Surface Methodology that determined 38.11 °C, 6.89 pH, 19.85 h and 179.15 rpm as optimum process parameters could improve the isolate L-asparaginase productivity. Maximum production of about 8 IU/ml/h was obtained with 0.4% dextrose, 0.1% yeast extract and 10 mM magnesium sulphate. In conclusion L-asparaginase of the recovered Stenotrophomonas maltophilia EMCC2297 isolate has characters enabling it to be used for medical therapeutic application.
Project description:l-Asparaginase (EC 184.108.40.206) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.
Project description:This study describes the production of native l-asparaginases by submerged fermentation from Aspergillus strains and provides the biochemical characterization, kinetic and thermodynamic parameters of the three ones that stood out for high l-asparaginase production. For comparison, the commercial fungal l-asparaginase was also studied. Both commercial and l-asparaginase from Aspergillus oryzae CCT 3940 showed optimum activity and stability in the pH range from 5 to 8 and the asparaginase from Aspergillus niger LBA 02 was stable in a more alkaline pH range. About the kinetic parameters, the denaturation constant increased with the heating temperature for all l-asparaginases, indicating that the l-asparaginase activity decreased at higher temperatures, especially above 60 °C. Moreover, l-asparaginase from A. oryzae CCT 3940 remained stable after 60 min at 50 °C. None of the l-asparaginases were inhibited by high NaCl concentrations, which are highly desirable for food industry application. The catalytic activities of all the l-asparaginases were enhanced by the presence of Mn2+ and inhibited by p-chloromercuribenzoate and iodoacetamide. The l-asparaginase from the Aspergillus strains and the commercial enzyme had similar K m when l-asparagine was used as substrate. None of the l-asparaginases, except the l-asparaginase from A. niger LBA 02, could hydrolyze the substrate l-glutamine, which is of interest for medical proposes, since the glutaminase activity is usually related to adverse reaction during the leukemia treatment. This study showed that these new three non-recombinant l-asparaginases studied have potential application in the food and pharmaceutical industries, especially due to their good thermostability.
Project description:Many side effects of current FDA-approved L-asparaginases have been related to their secondary L-glutaminase activity. The Wolinella succinogenes L-asparaginase (WoA) has been reported to be L-glutaminase free, suggesting it would have fewer side effects. Unexpectedly, the WoA variant with a proline at position 121 (WoA-P121) was found to have L-glutaminase activity in contrast to Uniprot entry P50286 (WoA-S121) that has a serine residue at this position. Towards understanding how this residue impacts the L-glutaminase property, kinetic analysis was coupled with crystal structure determination of these WoA variants. WoA-S121 was confirmed to have much lower L-glutaminase activity than WoA-P121, yet both showed comparable L-asparaginase activity. Structures of the WoA variants in complex with L-aspartic acid versus L-glutamic acid provide insights into their differential substrate selectivity. Structural analysis suggests a mechanism by which residue 121 impacts the conformation of the conserved tyrosine 27, a component of the catalytically-important flexible N-terminal loop. Surprisingly, we could fully model this loop in either its open or closed conformations, revealing the roles of specific residues of an evolutionary conserved motif among this L-asparaginase family. Together, this work showcases critical residues that influence the ability of the flexible N-terminal loop for adopting its active conformation, thereby effecting substrate specificity.
Project description:We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.