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1.
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. 相似文献
2.
Asparagine synthetase B (AsnB) catalyzes the formation of asparagine in an ATP-dependent reaction using glutamine or ammonia as a nitrogen source. To obtain a better understanding of the catalytic mechanism of this enzyme, we report the cloning, expression, and kinetic analysis of the glutamine- and ammonia-dependent activities of AsnB from Vibrio cholerae. Initial velocity, product inhibition, and dead-end inhibition studies were utilized in the construction of a model for the kinetic mechanism of the ammonia- and glutamine-dependent activities. The reaction sequence begins with the ordered addition of ATP and aspartate. Pyrophosphate is released, followed by the addition of ammonia and the release of asparagine and AMP. Glutamine is simultaneously hydrolyzed at a second site and the ammonia intermediate diffuses through an interdomain protein tunnel from the site of production to the site of utilization. The data were also consistent with the dead-end binding of asparagine to the glutamine binding site and PP(i) with free enzyme. The rate of hydrolysis of glutamine is largely independent of the activation of aspartate and thus the reaction rates at the two active sites are essentially uncoupled from one another. 相似文献
3.
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. 相似文献
4.
Fine structure analysis of the Chinese hamster AS gene encoding asparagine synthetase 总被引:3,自引:0,他引:3
Overlapping cDNAs for Chinese hamster ovary (CHO) asparagine synthetase (AS) were isolated from a library prepared from an AS-overproducing cell line. The sequence was determined and shown to contain an open reading frame encoding a protein of Mr 64,300. The predicted amino acid sequence for the CHO AS enzyme was compared to that of the human AS enzyme and found to be 95% homologous. A potential glutamine amide transfer domain, with sequence similarity to amidotransferases from bacteria and yeast, was identified in the N-terminal portion of the protein. The cDNAs were used to screen a library of phage containing wild type CHO DNA and the genomic AS sequences were detected on three overlapping phages. Determination of the fine structural organization showed that the CHO AS gene spanned 19 kilobases and was composed of 12 exons, three of which contained the glutamine amidotransferase domain. The 5' flanking sequences were highly G + C-rich and, like other housekeeping genes, lacked TATA and CAAT boxes. 相似文献
5.
Douangamath A Walker M Beismann-Driemeyer S Vega-Fernandez MC Sterner R Wilmanns M 《Structure (London, England : 1993)》2002,10(2):185-193
Since reactive ammonia is not available under physiological conditions, glutamine is used as a source for the incorporation of nitrogen in a number of metabolic pathway intermediates. The heterodimeric ImGP synthase that links histidine and purine biosynthesis belongs to the family of glutamine amidotransferases in which the glutaminase activity is coupled with a subsequent synthase activity specific for each member of the enzyme family. Its X-ray structure from the hyperthermophile Thermotoga maritima shows that the glutaminase subunit is associated with the N-terminal face of the (beta alpha)(8) barrel cyclase subunit. The complex reveals a putative tunnel for the transfer of ammonia over a distance of 25 A. Although ammonia tunneling has been reported for glutamine amidotransferases, the ImGP synthase has evolved a novel mechanism, which extends the known functional properties of the versatile (beta alpha)(8) barrel fold. 相似文献
6.
Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization. 相似文献
7.
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. 相似文献
8.
9.
Asparagine synthetases of Klebsiella aerogenes: properties and regulation of synthesis 总被引:3,自引:1,他引:2
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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. 相似文献
10.
Bellucci Michele Ederli Luisa De Marchis Francesca Pasqualini Stefania Arcioni Sergio 《Plant Cell, Tissue and Organ Culture》2004,78(2):139-150
Asparagine and glutamine are major forms of nitrogen in the phloem sap of many higher plants. In vascular plants, glutamine-dependent asparagine synthetase (AS) is the primary source of asparagine. In Escherichia coli, asparagine is synthesized by the action of two distinct enzymes, AS-A which utilizes ammonia as a nitrogen donor, and AS-B which utilizes both glutamine and ammonia as substrates, but with a preference for glutamine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim to introduce a new ammonium assimilation pathway in plants. The bacterial gene is placed under the control of light-dependent promoters, and introduced by transformation into Lotus corniculatus plants. Analysis of transgenic plants has revealed a phenomenon of transgene silencing which has prevented asnA expression in several transgenics. The asnA-expressing plants are characterized by premature flowering and reduced growth. A significant reduction of the total free amino acid accumulation in transgenic plants is observed. Surprisingly, the content of asparagine in wild-type plants is about 2.5-fold higher than that of transgenic plants. While glutamine levels in transgenic plants are about 3–4-fold higher than those in wild-type plants, aspartate levels are significantly lower. Transformation with asnA also induced a significant reduction of photosynthesis when measured under saturated light and ambient CO2 conditions. 相似文献
11.
Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase
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A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites. 相似文献
12.
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. 相似文献
13.
Reetika Manhas Pankaj Tripathi Sameena Khan Bhavana Sethu Lakshmi Shambhu Krishan Lal Venkatraman Subramanian Gowri Amit Sharma Rentala Madhubala 《The Journal of biological chemistry》2014,289(17):12096-12108
Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target. 相似文献
14.
Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation. 相似文献
15.
16.
Davydova M. A. Bruskova R. K. Gazizov I. S. Izmailov S. F. 《Russian Journal of Plant Physiology》2002,49(5):614-621
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. 相似文献
17.
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. 相似文献
18.
Esmeralda Parra-Peralbo Manuel Pineda Miguel Aguilar 《Molecular biology reports》2009,36(8):2249-2258
A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net
charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent
AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity
to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light. 相似文献
19.
NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation. NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers. 相似文献
20.
Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana. 总被引:15,自引:3,他引:12
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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. 相似文献