L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase.

An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression.     

Role of asparaginase variable loop at the carboxyl terminal of the alpha subunit in the determination of substrate preference in plants

Structural determinants responsible for the substrate preference of the potassium-independent (ASPGA1) and -dependent (ASPGB1) asparaginases from Arabidopsis thaliana have been investigated. Like ASPGA1, ASPGB1 was found to be catalytically active with both l-Asn and β-Asp-His as substrates, contrary to a previous report. However, ASPGB1 had a 45-fold higher specific activity with Asn as substrate than ASPGA1. A divergent sequence between the two enzymes forms a variable loop at the C-terminal of the alpha subunit. The results of dynamic simulations have previously implicated a movement of the C-terminus in the allosteric transduction of K⁺-binding at the surface of LjNSE1 asparaginase. In the crystal structure of Lupinus luteus asparaginase, most residues in this segment cannot be visualized due to a weak electron density. Exchanging the variable loop in ASPGA1 with that from ASPGB1 increased the affinity for Asn, with a 320-fold reduction in K _m value. Homology modeling identified a residue specific to ASPGB1, Phe¹⁶², preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly²⁴⁶, a residue participating in an oxyanion hole which stabilizes a negative charge forming on the side chain oxygen of asparagine during catalysis. Replacement with the corresponding leucine from ASPGA1 specifically lowered the V _max value with Asn as substrate by 8.4-fold.     

Two Methyl Jasmonate-Insensitive Mutants Show Altered Expression of AtVsp in Response to Methyl Jasmonate and Wounding

Jasmonates are plant signal molecules that are derived from lipids through the action of lipoxygenase. Jasmonates regulate gene expression during plant development and in response to water deficit, wounding, and pathogen elicitors. The signal transduction chain that mediates jasmonate action was investigated by isolating and studying two methyl jasmonate (MeJA)-insensitive mutants of Arabidopsis thaliana. The recessive mutants, jin1 and jin4, are nonallelic and neither corresponds to coi1, a previously identified MeJA-insensitive mutant. Both mutants showed reduced sensitivity to MeJA-mediated root growth inhibition as well as reduced MeJA induction of AtVsp in leaves. Expression of AtVsp in flowers was not altered in the mutants. Furthermore, MeJA modulation of the jasmonate-responsive lipoxygenase and phenylalanine ammonia lyase genes was not altered in the mutants. jin4 plants exhibited increased sensitivity to abscisic acid in seed germination assays, whereas jin1 plants showed wild-type sensitivity. Neither mutant showed altered sensitivity to ethylene in hypocotyl growth inhibition assays. jin1 and jin4 identify genes that modulate the response of AtVsp to MeJA in leaves of A. thaliana.     

Identification of a new locus in the Escherichia coli cotransduction gap that represents a new genetic component of the L-asparagine utilization system

A temperature-sensitive Escherichia coli mutant defective for the ability to utilize L-asparagine as a sole nitrogen source was isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutation (asu) produces two distinct phenotypic effects. Mutant strains grow poorly at high temperature on minimal plates containing asparagine as the sole nitrogen source; this effect is greatly exacerbated by the presence of methionine. Mutant strains utilize L-asparagine as a nitrogen source three to four times more efficiently at permissive temperatures than the wild-type strains. The mutation maps at 32.4 min on the E. coli chromosome, within the E. coli cotransduction gap. Mutant strains produce normal amounts of thermo-stable L-asparaginase I activity. The mutation therefore affects a component of the asparagine utilization system other than the catabolism of asparagine within the cell; it probably affects asparagine uptake.     

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg²⁺. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn''t affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.     

L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an externally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibition, whereas the internal activity is highly inhibited by this compound.     

Genetic and physiological relationships between L-asparaginase I and asparaginase II in Saccharomyces cerevisiae.

The cistron that codes for L-asparaginase I in Saccharomyces cerevisiae (aspl) is not genetically linked to either of the cistrons coding for expression of asparaginase II (asp2 and asp3). Cells containing different combinations of theses enzymes grow at different rates in media in which L-asparagine or D-asparagine is the only source of nitrogen for cell replication. Cells lacking L-asparaginase I but possessing asparaginase II grow more rapidly in medium containing D-asparagine as a nitrogen source than cells containing both enzymes, even though D-asparagine is not a substrate of L-asparaginase I. These results indicate that L-asparaginase I and asparaginase II interact in some way to regulate the utilization of asparagine as a nitrogen source for cell growth.     

Asparagine utilization in Escherichia coli

Asparagine-requiring auxotrophs of Escherichia coli K-12 that have an active cytoplasmic asparaginase do not conserve asparagine supplements for use in protein synthesis. Asparagine molecules entering the cell in excess of the pool required for use of this amino acid in protein synthesis are rapidly degraded rather than accumulated. Supplements are conserved when asparagine degradation is inhibited by the asparagine analogue 5-diazo-4-oxo-l-norvaline (DONV) or mutation to cytoplasmic asparaginase deficiency. A strain deficient in cytoplasmic asparaginase required approximately 260 mumol of asparagine for the synthesis of 1 g of cellular protein. The cytoplasmic asparaginase (asparaginase I) is required for growth of cells when asparagine is the nitrogen source. This enzyme has an apparent K(m) for l-asparagine of 3.5 mM, and asparaginase activity is competitively inhibited by DONV with an apparent K(i) of 2 mM. The analogue provides a time-dependent, irreversible inhibition of cytoplasmic asparaginase activity in the absence of asparagine.     

Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli L-asparaginase I

AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is positively cooperative, displaying a sigmoidal substrate dependence curve with an [S](0.5) of 1 mM L-asparagine and a Hill coefficient (n(H)) of 2.6. Binding of L-asparagine to an allosteric site was observed in the crystal structure concomitant with a reorganization of the quarternary structure, relative to the apo enzyme. The carboxyl group of the bound asparagine makes salt bridges and hydrogen bonds to Arg240, while the N(delta2) nitrogen interacts with Thr162. Mutation of Arg240 to Ala increases the [S](0.5) value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity. Mutation of Thr162 to Ala results in an active enzyme with no cooperativity. Transmission of the signal from the allosteric site to the active site appears to involve subtle interactions at the dimer-dimer interface and relocation of Gln118 into the vicinity of the active site to position the probable catalytic water molecule. These data define the structural basis for the cooperative regulation of the intracellular asparaginase that is required for proper functioning within the cell.     

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

Asparagine metabolism and asparaginase activity in a euryhaline Chlamydomonas species.

A Chlamydomonas species isolated from a marine environment possesses an L-asparaginase, an enzyme not yet reported in the microalgae. This enzyme enabled the organism to grow as well with asparagine as sole nitrogen source as with inorganic nitrogen sources (NO3-, NH4+). Only the amide nitrogen was used for growth since growth did not occur on aspartate and aspartate accumulated in the media when cells were either grown on asparagine or during short-term incubations with L-[U-14C]asparagine. Cells grown on NO3-, NH4+, or L-asparagine in batch culture possessed equivalent asparaginase activities. However, nitrogen-limited cells possessed four times the activity of cells grown with sufficient nitrogen for normal growth, regardless of the possessed the lowest activity per cell, while lag phase and stationary phase cells possessed greater activity. The enzyme behaved like a periplasmic space enzyme since (1) breaking the cells did not release into solution more activity than was shown by whole cells and (2) whole cells converted L-[U-14C]asparagine to [14C]aspartate with little intracellular accumulation of radioactivity. Cell-free preparations of the enzyme possessed a Km value for asparagine of 1.1 x 10-4 M, with no glutaminase activity.     

Overexpression of the aspartic protease ASPG1 gene confers drought avoidance in Arabidopsis

Drought is one of the most severe environmental stresses affecting plant growth and limiting crop production. Although many genes involved in adaptation to drought stress have been disclosed, the relevant molecular mechanisms are far from understood. This study describes an Arabidopsis gene, ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1), that may function in drought avoidance through abscisic acid (ABA) signalling in guard cells. Overexpression of the ASPG1 gene enhanced ABA sensitivity in guard cells and reduced water loss in ectopically overexpressing ASPG1 (ASPG1-OE) transgenic plants. In ASPG1-OE plants, some downstream targets in ABA and/or drought-signalling pathways were altered at various levels, suggesting the involvement of ASPG1 in ABA-dependent drought avoidance in Arabidopsis. By analysing the activities of several antioxidases including superoxide dismutase and catalase in ASPG1-OE plants, the existence was demonstrated of an effective detoxification system for drought avoidance in these plants. Analysis of ProASPG1-GUS lines showed a predominant guard cell expression pattern in various aerial tissues. Moreover, the protease activity of ASPG1 was characterized in vitro, and two aspartic acid sites, D180 and D379, were found to be key residues for ASPG1 aspartic protease activity in response to ABA. In summary, these findings suggest that functional ASPG1 may be involved in ABA-dependent responsiveness and that overexpression of the ASPG1 gene can confer drought avoidance in Arabidopsis.     

Characterization of two forms of asparaginase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae X2180-1A synthesizes two forms of asparaginase: L-asparaginase I, an internal constitutive enzyme, and asparaginase II, an external enzyme which is secreted in response to nitrogen starvation. The two enzymes are biochemically and genetically distinct. The structural gene for asparaginase I (asp 1) is closely linked to the trp 4 gene on chromosome IV. The gene controlling the synthesis of asparaginase II is not linked to either the trp 4 or asp 1 genes. The rate of biosynthesis of asparaginase II is unaltered in yeast strains carrying the structural gene mutation for asparaginase I. Asparaginase II has been purified approximately 300-fold from crude extracts of Saccharomyces by heat and pH treatment, ethanol fractionation, ammonium sulfate fractionation followed by Sephadex G-25 chromatography, and DEAE-cellulose chromatography. Multiple activity peaks were obtained which, upon gas chromatographic analysis, exhibit varying mannose to protein ratios. Asparaginase I has been purified approximately 100-fold from crude extracts of Saccharomyces by protamine sulfate treatment, ammonium sulfate fractionation, gel permeation chromatography, and DEAE-cellulose chromatography. No carbohydrate component was observed upon gas chromatographic analysis. Comparative kinetic and analytic studies show the two enzymes have little in common except their ability to hydrolyze L-asparagine to L-aspartic acid and ammonia.     

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.     

Interactions between abscisic acid and ethylene signaling cascades

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.     

L-Asparagainases from Citrobacter freundii.

Three enzymes which catalyze the hydrolysis of L-asparagine have been identified in extracts of Citrobacter freundii. One of these (asparaginase-glutaminase (EC 3.5.1.1) also shows substantial glutaminase activity. This enzyme is extremely labile, is sensitive to inactivation by p-chloromercuribenzoate, and is not protected by dithiothreitol. A second enzyme (asparaginase B) is also sensitive to mercurials but is protected from inactivation by dithiothreitol. This enzyme has a relatively low affinity for L-asparagine (Km = 1.7-10(-3) M). The third enzyme (asparaginase A) is insensitive to inactivation by mercurials, is stable upon long term storage and has a relatively high affinity for L-asparagine (Km = 2.9-10(-5) M). This enzyme has been purified to homogeneity and has a molecular weight of approx. 140 000; the subunit weight being approx. 33 000. The C. freundii asparaginase A produced significant increases in the survival time of C3H/HE mice carrying the 6C3HED lymphoma tumor.