Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.
ABSTRACT: 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: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.
Project description:L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types. Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers.
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:This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme.
Project description:Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.
Project description:L-Asparaginase (L-asparagine aminohydrolase, E.C. 220.127.116.11) 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.