Expression of human asparagine synthetase in Saccharomyces cerevisiae

Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae. The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell. Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed. The yeast expression plasmid was constructed for optimal production of the recombinant enzyme. In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced. This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity.     

Asparagine synthetases of Klebsiella aerogenes: properties and regulation of synthesis

We isolated pleiotropic mutants of Klebsiella aerogenes with the transposon Tn5 which were unable to utilize a variety of poor sources of nitrogen. The mutation responsible was shown to be in the asnB gene, one of two genes coding for an asparagine synthetase. Mutations in both asnA and asnB were necessary to produce an asparagine requirement. Assays which could distinguish the two asparagine synthetase activities were developed in strains missing a high-affinity asparaginase. The asnA and asnB genes coded for ammonia-dependent and glutamine-dependent asparagine synthetases, respectively. Asparagine repressed both enzymes. When growth was nitrogen limited, the level of the ammonia-dependent enzyme was low and that of the glutamine-dependent enzyme was high. The reverse was true in a nitrogen-rich (ammonia-containing) medium. Furthermore, mutations in the glnG protein, a regulatory component of the nitrogen assimilatory system, increased the level of the ammonia-dependent enzyme. The glutamine-dependent asparagine synthetase was purified to 95%. It was a tetramer with four equal 57,000-dalton subunits and catalyzed the stoichiometric generation of asparagine, AMP, and inorganic pyrophosphate from aspartate, ATP, and glutamine. High levels of ammonium chloride (50 mM) could replace glutamine. The purified enzyme exhibited a substrate-independent glutaminase activity which was probably an artifact of purification. The tetramer could be dissociated; the monomer possessed the high ammonia-dependent activity and the glutaminase activity, but not the glutamine-dependent activity. In contrast, the purified ammonia-dependent asparagine synthetase, about 40% pure, had a molecular weight of 80,000 and is probably a dimer of identical subunits. Asparagine inhibited both enzymes. Kinetic constants and the effect of pH, substrate, and product analogs were determined. The regulation and biochemistry of the asparagine synthetases prove the hypothesis strongly suggested by the genetic and physiological evidence that a glutamine-dependent enzyme is essential for asparagine synthesis when the nitrogen source is growth rate limiting.     

A convenient gHMQC-based NMR assay for investigating ammonia channeling in glutamine-dependent amidotransferases: studies of Escherichia coli asparagine synthetase B

X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformation have revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels, giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficiently into a separate synthetase site where it undergoes further reaction. We now report a very convenient isotope-edited 1H NMR-based assay that can be used to probe the transfer of ammonia between the active sites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B (AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solution can react with the thioester intermediate formed during the glutaminase half-reaction by accessing the N-terminal active site of AS-B during catalytic turnover. These observations are consistent with a model in which exogenous ammonia can access the intramolecular tunnel in AS-B during glutamine-dependent asparagine synthesis, in contrast to expectations based on studies of class I amidotransferases.     

Bovine pancreatic asparagine synthetase explored with substrate analogs and specific monoclonal antibodies.

Several substrate analogs were tested for their ability to inhibit bovine pancreatic asparagine synthetase. Of the substrate analogs tested both 6-diazo-5-oxo-L-norleucine (DON) and 5-chloro-4-oxo-L-norvaline (CONV) were shown to inhibit the enzyme strongly. DON inhibited the glutaminase and glutamine-dependent asparagine synthetase activities and CONV inhibited the ammonia-dependent activity as well. Both of these inhibitors appeared to be relatively tight binding since desalting failed to remove the inhibition. The inactivation of bovine pancreatic asparagine synthetase by DON is accompanied by a shift from a 47,000 molecular weight monomer to a 96,000 molecular weight dimer as observed by HPLC gel filtration chromatography. This DON-induced shift is prevented by the presence of the substrate glutamine. A monoclonal antibody known to inhibit specifically the ammonia-dependent and glutamine-dependent asparagine synthetase activities but not glutaminase (monoclonal antibody 2B4) binds to both the monomer and the dimer forms of untreated enzyme, as well as to the dimer form of the DON-inactivated enzyme. On the other hand, a monoclonal antibody known to inhibit specifically the glutaminase and glutamine-dependent activities and not the ammonia-dependent asparagine synthetase (monoclonal antibody 5A6) binds to both forms of untreated enzyme but cannot bind to the DON-inactivated enzyme. These data are used to describe the relation of regions of the active site of asparagine synthetase in relation to antibody binding sites.     

Expression of human asparagine synthetase in Escherichia coli

Human asparagine synthetase was expressed in Escherichia coli. Synthesis of the enzyme was demonstrated by immunoblotting and by complementation of asparagine auxotrophy in E. coli. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. Compared to asparagine synthetase isolated from beef pancreas, the one expressed in E. coli migrated at a slightly slower rate on a denaturing protein gel. In contrast with previous reports, the data obtained here strongly suggest that the active enzyme is a homodimer. The production of soluble and active enzyme was shown to be highly temperature-dependent. Expression at 37 degrees C yielded no soluble enzyme, whereas growth at 30 and 21 degrees C favored the production of soluble asparagine synthetase. The incubation temperature was also important for complementation of asparagine auxotrophy in E. coli, as growth in the absence of asparagine occurred at 30 degrees C and not at 37 degrees C.     

Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli

Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.     

Assimilation of Ammonium Nitrogen by Heterotrophic and Symbiotrophic White Lupine Plants

The activities of glutamate dehydrogenase, asparagine synthetase, and total glutamine synthetase in the organs of the white lupine (Lupinus albus L.) plants were measured during plant growth and development. In addition, the dynamics of free amino acids and amides in plant organs was followed. It was shown that the change in the nutrition type was important for controlling enzyme activities in the organs examined and, consequently, for directing the pathway of ammonium nitrogen assimilation. As long as the plants remained heterotrophic, glutamine-dependent asparagine synthetase of cotyledons and glutamine synthetase of leaves apparently played a major role in the assimilation of ammonium nitrogen. In symbiotrophic plants, root nodules became an exclusive site of asparagine synthesis, and the role of leaf glutamine synthetase increased. Unlike glutamine synthetase and asparagine synthetase, glutamate dehydrogenase activity was present in all organs examined and was less dependent on the nutrition type. This was also indicated by a weak correlation of glutamate dehydrogenase activity with the dynamics of free amino acid and amide content in these organs. It is supposed that glutamine synthetase plays a leading role in both the primary assimilation of ammonium, produced during symbiotic fixation of molecular nitrogen in root nodules, and in its secondary assimilation in cotyledons and leaves. On the other hand, secondary nitrogen assimilation in the axial organs occurs via an alternative glutamate dehydrogenase pathway.     

The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln

The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn). This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA(Gln) and Asn-tRNA(Asn) synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/K(m) of 1368.4 s(-1)/mM and 3059.3 s(-1)/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys(52), Ser(128), Ser(152)) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA(Asn) and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA(Asn) by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.     

A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production

The Corynebacterium glutamicum mutant KY9714, originally isolated as a lysozyme-sensitive mutant, does not grow at 37 degrees C. Complementation tests and DNA sequencing analysis revealed that a mutation in a single gene of 1,920 bp, ltsA (lysozyme and temperature sensitive), was responsible for its lysozyme sensitivity and temperature sensitivity. The ltsA gene encodes a protein homologous to the glutamine-dependent asparagine synthetases of various organisms, but it could not rescue the asparagine auxotrophy of an Escherichia coli asnA asnB double mutant. Replacement of the N-terminal Cys residue (which is conserved in glutamine-dependent amidotransferases and is essential for enzyme activity) by an Ala residue resulted in the loss of complementation in C. glutamicum. The mutant ltsA gene has an amber mutation, and the disruption of the ltsA gene caused lysozyme and temperature sensitivity similar to that in the KY9714 mutant. L-Glutamate production was induced by elevating growth temperature in the disruptant. These results indicate that the ltsA gene encodes a novel glutamine-dependent amidotransferase that is involved in the mechanisms of formation of rigid cell wall structure and in the L-glutamate production of C. glutamicum.     

The topology of the glutamine and ATP binding sites of human asparagine synthetase.

Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies. The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase. The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP). The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis. Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity. A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers. It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains. These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.     

Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [¹⁴C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [¹⁴C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.

Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

     

An alternative mechanism for the nitrogen transfer reaction in asparagine synthetase.

In the absence of crystallographic data, the mechanism of nitrogen transfer from glutamine in asparagine synthetase (AS) remains under active investigation. Surprisingly, the glutamine-dependent AS from Escherichia coli (AsnB) appears to lack a conserved histidine residue, necessary for nitrogen transfer if the reaction proceeds by the accepted pathway in other glutamine amidotransferases, but retains the ability to synthesize asparagine. We propose an alternative mechanism for nitrogen transfer in AsnB which obviates the requirement for participation of histidine in this step. Our hypothesis may also be more generally applicable to other glutamine-dependent amidotransferases.     

Amino acid sequence of a peptide containing an essential cysteine residue of Escherichia coli GMP synthetase.

The amino acid sequence of a 51-residue tryptic peptide of citraconylated [1-14C]carboxamidomethyl-labeled Escherichia coli GMP synthetase was determined by sequenator analyses of the intact peptide and fragments obtained by cleavage of the peptide with cyanogen bromide, trypsin, and Staphylcoccus aureus strain V8 protease. The cysteine residue of this peptide fragment is essential for glutamine-dependent GMP synthesis activity and is implicated in formation of a hypothetical covalent glutamyl-enzyme intermediate. There is essentially cysteine-containing regions of two other glutamine amidotransferases, Pseudomonas putida anthranilate synthetase Component II and chicken liver formylglycinamide ribonucleotide amidotransferase. There is, however, a cluster of amino acids with "antipathy" for helix formation and a "nonessential" cysteine of anthranilate synthetase Component II.     

Purification and characterization of beef pancreatic asparagine synthetase

Bovine pancreatic asparagine synthetase has been partially purified using ammonium sulfate fractionation, DEAE ion-exchange, Cibacron Blue affinity chromatography, and HPLC anion-exchange chromatography to a specific activity of 170 nmol asparagine produced min-1 mg protein-1, or 1400-fold, from a crude homogenate. Using HPLC size exclusion chromatography, an apparent molecular weight of 110,000-120,000 was determined. An aspartyl-adenylate intermediate was found to occur by demonstrating an 18O transfer from [18O]Asp to AMP that was detected with 31P NMR. A number of divalent metals were found to be able to replace magnesium with retention of activity, but none produced as high an activity as Mg2+, and the stoichiometry of the ATP/Mg2+ ratio was found to be 1. The chloride ion was found to stimulate the glutamine-dependent and glutaminase reactions, but the ammonia-dependent reaction was inhibited. Chloride appeared to be a competitive inhibitor with respect to ammonia and produced negative cooperativity.     

Kinetic mechanism of beef pancreatic L-asparagine synthetase

The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.     

Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.     

Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B

Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.     

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.