A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

A systematic review of recent trends in research on therapeutically significant L-asparaginase and acute lymphoblastic leukemia.

L-asparaginases are mostly obtained from bacterial sources for their application in the therapy and food industry. Bacterial L-asparaginases are employed in the treatment of Acute Lymphoblastic Leukemia (ALL) and its subtypes, a type of blood and bone marrow cancer that results in the overproduction of immature blood cells. It also plays a role in the food industry in reducing the acrylamide formed during baking, roasting, and frying starchy foods. This importance of the enzyme makes it to be of constant interest to the researchers to isolate novel sources. Presently L-asparaginases from E. coli native and PEGylated form, Dickeya chrysanthemi (Erwinia chrysanthemi) are in the treatment regime. In therapy, the intrinsic glutaminase activity of the enzyme is a major drawback as the patients in treatment experience side effects like fever, skin rashes, anaphylaxis, pancreatitis, steatosis in the liver, and many complications. Its significance in the food industry in mitigating acrylamide is also a major reason. Acrylamide, a potent carcinogen was formed when treating starchy foods at higher temperatures. Acrylamide content in food was analyzed and pre-treatment was considered a valuable option. Immobilization of the enzyme is an advancing and promising technique in the effective delivery of the enzyme than in free form. The concept of machine learning by employing the Artificial Network and Genetic Algorithm has paved the way to optimize the production of L-asparaginase from its sources. Gene-editing tools are gaining momentum in the study of several diseases and this review focuses on the CRISPR-Cas9 gene-editing tool in ALL.

Can recombinant technology address asparaginase Erwinia chrysanthemi shortages?

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Bacterial L-asparaginase has played an important role in ALL treatment for several decades; however, hypersensitivity reactions to Escherichia coli-derived asparaginases often preclude their use. Inability to receive asparaginase due to hypersensitivities is associated with poor patient outcomes. Erwinia chrysanthemi-derived asparaginase (ERW) is an effective, non-cross-reactive treatment option, but is limited in supply. Consequently, alternative asparaginase preparations are needed to ensure asparaginase availability for patients with hypersensitivities. Recombinant technology can potentially address this unmet need by programming cells to produce recombinant asparaginase. JZP-458, a recombinant Erwinia asparaginase derived from a novel Pseudomonas fluorescens expression platform with no immunologic cross-reactivity to E. coli-derived asparaginases, has the same primary amino acid sequence as ERW, with comparable activity based on in vitro measurements. The efficient manufacturing of JZP-458 would provide an additional asparaginase preparation for patients with hypersensitivities.

Determination of extent of PEGylation using denaturing capillary isoelectric focussing.

Conjugated proteins and enzymes are often formed using N-hydroxysuccinimide (NHS) chemistry, which reacts with free primary amines resulting in a loss of charge and a reduction in isoelectric point (pI). Measurement of the extent of reaction of these conjugates is critical for biopharmaceutical developers. Due to this change in protein charge state, denaturing capillary isoelectric focussing (cIEF) offers a potentially straightforward and convenient approach for extent-of-reaction quantification. Here, we demonstrate the potential of this technique with poly(ethylene glycol) (PEG) conjugates of Erwinia chrysanthemil-asparaginase (ErA). Development of an appropriate sample preparation technique is critical to achieving reproducible cIEF electropherograms, particularly for denaturation-resistant proteins such as ErA, and an emphasis was placed on this during development of the PEG-ErA cIEF method. cIEF electropherograms demonstrating a distribution of PEGylation states in a bell-shaped curve were obtained, and assignment of PEGylation states to these peaks was critical to routine use of the method. The method is sensitive enough to resolve non-lysine adducts of PEG (such as those conjugated to histidine residues) and was shown to give reproducible results over a 2 year period. Biopharmaceutical developers should consider cIEF for extent of reaction monitoring and measurement for conjugates of free amine groups.

Mechanism of Catalysis by l-Asparaginase.

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.

Acidic isoforms of Erwinase form part of the product: Correlation with clinical experience.

Erwinia chrysanthemil-asparaginase (ErA) has been used for the treatment of acute lymphoblastic leukaemia (ALL) for decades, and its safety and efficacy have been well demonstrated. ErA drug substance and drug product contain a small proportion of acidic isoforms, with a known mechanism of formation, which have been shown to be minor conformational variants retaining enzymatic activity and function. Specifications for these acidic isoforms were set with an extremely limited data set, and with further manufacturing experience, it can now be demonstrated that they were set too tightly. Here, we consider the ability of the manufacturing process to meet the current acidic isoforms specifications, as well as clinical outcomes from drug product containing a higher proportion of isoforms. Compared with the historical clinical experience with the drug, there appeared to be no difference in the rate of adverse event reporting (e.g., hypersensitivity or other events) when drug product with relatively higher acidic isoforms was administered. ErA acidic isoforms comprise part of the ErA product and appear to have no clinical relevance, so a realignment of process capability and specification may be warranted. Biopharmaceutical developers should exercise caution when setting specifications with limited data, to avoid process capability pitfalls later.

A comprehensive review on microbial l-asparaginase: Bioprocessing, characterization, and industrial applications.

l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein synthesis in cancer cells, making l-asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l-Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l-asparaginase leads to the reduction of acrylamide formation in fried food items. l-Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l-asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long-term clinical use of l-asparaginase. Present review provides thorough information on microbial l-asparaginase bioprocess optimization including submerged fermentation and solid-state fermentation for l-asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l-asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l-asparaginase with less adverse effects.

Decreasing the immunogenicity of Erwinia chrysanthemi asparaginase via protein engineering: computational approach.

Immunogenicity of therapeutic proteins is one of the main challenges in disease treatment. L-Asparaginase is an important enzyme in cancer treatment which sometimes leads to undesirable side effects such as immunogenic or allergic responses. Here, to decrease Erwinase (Erwinia chrysanthemiL-Asparaginase) immunogenicity, which is the main drawback of the enzyme, firstly conformational B cell epitopes of Erwinase were predicted from three-dimensional structure by three different computational methods. A few residues were defined as candidates for reducing immunogenicity of the protein by point mutation. In addition to immunogenicity and hydrophobicity, stability and binding energy of mutants were also analyzed computationally. In order to evaluate the stability of the best mutant, molecular dynamics simulation was performed. Among mutants, H240A and Q239A presented significant reduction in immunogenicity. In contrast, the immunogenicity scores of D235A slightly decreased according to two servers. Binding affinity of substrate to the active site reduced significantly in K265A and E268A. The final results of molecular dynamics simulation indicated that H240A mutation has not changed the stability, flexibility, and the total structure of desired protein. Overall, point mutation can be used for reducing immunogenicity of therapeutic proteins, in this context, in silico approaches can be used to screen suitable mutants.

PEGylated E. coli asparaginase desensitization: an effective and feasible option for pediatric patients with acute lymphoblastic leukemia who have developed hypersensitivity to pegaspargase in the absence of asparaginase Erwinia chrysanthemi availability.

Asparaginase is an important component of multi-agent chemotherapy for the treatment of pediatric acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LLy). Hypersensitivity to the PEGylated form, pegaspargase, is the most common toxicity observed and is ideally addressed by substituting multiple doses of erwinia asparaginase for each subsequent dose of pegaspargase. An international shortage of erwinia asparaginase has limited the therapeutic options for those experiencing pegaspargase hypersensitivity. Here, we report pegaspargase can be safely administered, while maintaining sustained levels of asparaginase activity, to patients who have had a prior hypersensitivity reaction to pegaspargase by using a standard rapid desensitization protocol. Ten patients with prior hypersensitivity reactions to pegaspargase were treated by using a standardized rapid desensitization protocol. Eight patients had therapeutic asparaginase levels between days 4 and 7 of ≥0.05 IU/mL, and seven patients continued to have sustained levels above ≥0.1 IU/mL between days 10 and 14. Based on chemotherapy regimens, five of these patients successfully received more than one dose of pegaspargase utilizing this protocol.

Acylated sulfonamide adenosines as potent inhibitors of the adenylate-forming enzyme superfamily.

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Šcrystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.

L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression.

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.

Efficacy of Asparaginase Erwinia chrysanthemi With and Without Temozolomide Against Glioma Cells and Intracranial Mouse Medulloblastoma.

BACKGROUND: Anti-metabolites are less-myelosuppressive than DNA-damaging anticancer drugs and may be useful against brain tumors. MATERIALS AND METHODS: We evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas. RESULTS: Erwinaze treatment of cell lines and neurospheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (IC50s) of 0.12->10 IU/ml. Erwinaze at <1 IU/ml reduced temozolomide IC50s by 3.6- to 13-fold (300-1,200 µM to 40-330 µM). Seven-week-old SMO/SMO mice treated with Erwinaze (regardless of temozolomide treatment) had better survival 11 weeks post-therapy, compared to those not treated with Erwinaze (81.25% vs. 46.15, p=0.08). Temozolomide-treated mice developed 10% weight loss, impairing survival. All 16 mice treated with temozolomide (regardless of Erwinaze treatment) succumbed by 40-weeks of age, whereas 5/8 animals treated with Erwinaze alone and 2/6 controls survived (p=0.035). CONCLUSION: Erwinaze enhances cytotoxicity of temozolomide in vitro, and improves survival in SMO/SMO mice, likely by reducing cerebrospinal fluid glutamine. Temozolomide-associated toxicity prevented demonstration of any potential combinatorial advantage with Erwinaze in vivo.

Symptomatic Hyperammonemia With Erwinia chrysanthemi-derived Asparaginase in Pediatric Leukemia Patients.

Erwinia chrysanthemi-derived asparaginase is increasingly integral to acute lymphoblastic leukemia therapy. In our series, 16% of patients developed symptomatic hyperammonemia following Erwinia administration with symptoms including refractory nausea, vomiting, profound fatigue, malaise, and coma. This series of patients receiving Erwinia indicates higher than expected incidence of hyperammonemia, correlation between ammonia and asparaginase levels and therapeutic asparaginase activity levels despite dose reduction. The series provides evidence for investigation into which patients require intervention to prevent toxicity, which patients may have ammonia levels used as an asparaginase activity surrogate and which patients may achieve equivalent efficacy with abridged dosing.

Asparaginase Erwinia chrysanthemi effectively depletes plasma glutamine in adult patients with relapsed/refractory acute myeloid leukemia.

Depletion of glutamine (Gln) has emerged as a potential therapeutic approach in the treatment of acute myeloid leukemia (AML), as neoplastic cells require Gln for synthesis of cellular components essential for survival. Asparaginases deplete Gln, and asparaginase derived from Erwinia chrysanthemi (Erwinaze) appears to have the greatest glutaminase activity of the available asparaginases. In this Phase I study, we sought to determine the dose of Erwinaze that safely and effectively depletes plasma Gln levels to ≤ 120 µmol/L in patients with relapsed or refractory (R/R) AML. Five patients were enrolled before the study was halted due to issues with Erwinaze manufacturing supply. All patients received Erwinaze at a dose of 25,000 IU/m2 intravenously three times weekly for 2 weeks. Median trough plasma Gln level at 48 h after initial Erwinaze administration was 27.6 µmol/L, and 80% (lower limit of 1-sided 95% CI 34%) of patients achieved at least one undetectable plasma Gln value (< 12.5 µmol/L), with the fold reduction (FR) in Gln level at 3 days, relative to baseline, being 0.16 (p < 0.001 for rejecting FR = 1). No dose-limiting toxicities were identified. Two patients responded, one achieved partial remission and one achieved hematologic improvement after six doses of Erwinaze monotherapy. These data suggest asparaginase-induced Gln depletion may have an important role in the management of patients with AML, and support more pharmacologic and clinical studies on the mechanistically designed asparaginase combinations in AML.

Pharmacodynamic effects in the cerebrospinal fluid of rats after intravenous administration of different asparaginase formulations.

PURPOSE: Asparaginase (ASNase) is used to treat various hematological malignancies for its capacity to deplete asparagine (ASN) in serum and cerebrospinal fluid (CSF). Since the biological mechanisms underlying CSF asparagine depletion in humans are not yet fully elucidated, this study compared, for the first time, the pharmacological properties of three clinically used ASNase formulations in a rodent model. METHODS: Male Wistar rats were treated with E.coli-ASNase, PEG-ASNase, or ERW-ASNase at different doses. Serum and CSF amino-acid levels and ASNase activities were evaluated at 1 and 24 h after the intravenous administration of different ASNase doses. RESULTS: All the ASNase formulations showed higher activities in serum after 1 h than 24 h and completely deplete ASN. Mean ASNase activity in the CSF at 1 h was higher with ERW-ASNase compared to PEG-ASNase (36 ± 29 vs 8 ± 7 U/L, p < 0.037) and similar to E.coli-ASNase (21 ± 9 U/L, ns). ERW-ASNase and E.coli-ASNase at the highest doses were able to deplete ASN in the CSF after 1 h. This effect was transient and not evident at 24 h after treatment. CONCLUSIONS: Together with the ASN depletion in serum and CSF, a never before demonstrated transient penetration of ASNases into the CSF, more evident for non-pegylated formulations, was detected when the ASNases were administered at high dose.

Understanding the process-induced formation of minor conformational variants of Erwinia chrysanthemil-asparaginase.

During Erwinia chrysanthemil-asparaginase (ErA) manufacture, minor conformational variants are formed that elute in the acidic region of the analytical ion-exchange HPLC chromatogram. These variants retain enzymatic activity and form part of the biopharmaceutical product, but must be kept within acceptable limits through controlled operation of the manufacturing process. The high isoelectric point of the ErA native tetramer (8.6) leads to certain process steps being operated in the alkaline pH region. Previously, the formation of these species during processing was not fully understood. In this work, in-process samples were analysed, and alkaline pH (8-9) and hold time were found to be the governing parameters. Formation of ErA acidic species was found to be accelerated at higher pH values and longer hold times, suggesting potential control strategies for the manufacturing process. However, the kinetics of ErA conformational variant formation were found to be slow (0.15-0.25 percent per day at pH 8.5). Changes in the ErA melt temperature (Tm) with pH as determined by both differential scanning calorimetry and differential scanning fluorimetry were found to be predictive of the tendency to form the IEX-HPLC acidic species during processing. Biopharmaceutical process developers should be aware of such changes to proteins and build relevant control strategies into process validation plans.

In vitro screening and in silico validation revealed key microbes for higher production of significant therapeutic enzyme l-asparaginase.

l-asparaginase is an enzyme of medical prominence and reputable as a chemotherapeutic agent. It also has immense potential to cure autoimmune and infectious diseases. The vast application of this enzyme in healthcare sector increases its market demand. However, presently the huge market demand is not achieved completely. This serves the basis to explore better producer microbial strains to bridge the gap between huge demand and supply of this therapeutic enzyme. The present study deals with the successful screening of potent microorganisms producing l-asparaginase. 47 microorganisms were screened including bacteria, fungi, and yeasts. Among all, Penicillium lilacinum showed the highest enzyme activity i.e., 39.67 IU/ml. Shigella flexneri has 23.21 IU/ml of enzyme activity (highest among all the bacterial strain tested). Further, the 3-D structure of l-asparaginase from higher producer strains was developed and validated in silico for its activity. l-asparagine (substrate for l-asparaginase) was docked inside the binding pocket of P. lilacinum and S. flexneri. Docking score for the most common substrate l-asparagine is -6.188 (P. lilacinum), -5.576 (S. flexneri) which is quite good. Moreover, the chemical property of the binding pocket revealed that amino acid residues Phe 243, Gln 260, Gly 365, Asp 386 in P. lilacinum and residues Asp 181, Thr 318, Asn 320 in S. flexneri have an important role in H-bonding. The in silico results supports and strengthen the wet lab results. The outcome obtained motivates to take the present study result from lab to industry for the economic/massive production of this enzyme for the diverse therapeutic application.

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC.

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1ß3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1ß3GABAAR, is potentiated by 2-bromoethanol and ethanol. The results suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures and functional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.

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.

Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase.

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.