Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA^Asn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA^Asn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNA^Asn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.     

Cereal asparagine synthetase genes

Asparagine synthetase catalyses the transfer of an amino group from glutamine to aspartate to form glutamate and asparagine. The accumulation of free (nonprotein) asparagine in crops has implications for food safety because free asparagine is the precursor for acrylamide, a carcinogenic contaminant that forms during high‐temperature cooking and processing. Here we review publicly available genome data for asparagine synthetase genes from species of the Pooideae subfamily, including bread wheat and related wheat species (Triticum and Aegilops spp.), barley (Hordeum vulgare) and rye (Secale cereale) of the Triticeae tribe. Also from the Pooideae subfamily: brachypodium (Brachypodium dIstachyon) of the Brachypodiae tribe. More diverse species are also included, comprising sorghum (Sorghum bicolor) and maize (Zea mays) of the Panicoideae subfamily and rice (Oryza sativa) of the Ehrhartoideae subfamily. The asparagine synthetase gene families of the Triticeae species each comprise five genes per genome, with the genes assigned to four groups: 1, 2, 3 (subdivided into 3.1 and 3.2) and 4. Each species has a single gene per genome in each group, except that some bread wheat varieties (genomes AABBDD) and emmer wheat (Triticum dicoccoides; genomes AABB) lack a group 2 gene in the B genome. This raises questions about the ancestry of cultivated pasta wheat and the B genome donor of bread wheat, suggesting that the hybridisation event that gave rise to hexaploid bread wheat occurred more than once. In phylogenetic analyses, genes from the other species cluster with the Triticeae genes, but brachypodium, sorghum and maize lack a group 2 gene, while rice has only two genes, one group 3 and one group 4. This means that TaASN2, the most highly expressed asparagine synthetase gene in wheat grain, has no equivalent in maize, rice, sorghum or brachypodium. An evolutionary pathway is proposed in which a series of gene duplications gave rise to the five genes found in modern Triticeae species.     

Immunochemical characterization of rat testicular asparagine synthetase.

We studied immunochemical properties of rat testicular asparagine synthetase. Western blot analysis of testis extract with polyclonal antibody raised against purified asparagine synthetase revealed an immunoreactive band at 62 kDa. The pancreas, brain, thymus, and spleen also showed 62-kDa bands. The intensities of these bands were roughly proportional to the specific activities of the enzyme in these tissues. The antibody showed some degree of cross-reactivity to asparagine synthetases from human, beef, pig, mouse, guinea pig, chicken, and frog, but not carp. But the enzyme from human HL-60 cells and lower vertebrates reacted with the antibody less strongly than enzyme from rats. The N-terminal amino acid sequence of the enzyme, determined by the Edman degradation method, in 10 recovered residues was identical to that of human asparagine synthetase deduced from corresponding cDNA (I.L. Andrulis et al., 1987, Mol. Cell. Biol. 7, 2435-2443). Immunohistochemical staining of the testis showed the presence of asparagine synthetase mainly in Sertoli cells in the seminiferous tubules.     

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.     

A high-performance liquid chromatography assay for asparagine synthetase

A highly sensitive method for assaying asparagine synthetase and its glutaminase activity is presented. The amino acids L-asparagine, L-aspartate, L-glutamate, and L-glutamine, are separated by derivatization with o-phthaldialdehyde followed by reversed-phase high-performance liquid chromatography on an Altex ultrasphere-ODS C18 column. The elution is isocratic and the mobile phase used is 50 mM sodium acetate buffer (pH 5.9) with 30% methanol. This assay can easily detect picomoles of asparagine, which may be difficult to do with the other assays that have been described.     

Chloride affects the interaction between tyrosyl-tRNA synthetase and tRNA

The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg2+, chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration (< 200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened the PPi inhibition, the Ki(app)(PPi) values at 1 mM free Mg2+ were 140, 120, and 56 microM at 0, 50 and 150 mM KCl, respectively. Chloride weakened the AMP inhibition, the corresponding values for Ki(app)(AMP) were 0.35, 0.5, and 0.9 mM. The value of Km(app)(tRNA(Tyr)) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Best-fit analyses of the PPi inhibition, AMP inhibition and Km(app)(tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg2+ to the tRNA before the transfer reaction and a weakened binding of Mg2+ to the Tyr-tRNA-enzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg2+ is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg2+ from the aa-tRNA-enzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli.     

Overexpression and purification of asparagine synthetase from Escherichia coli.

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.     

Purification and properties of asparagine synthetase from rat liver.

Asparagine synthetase (L-aspartate:ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation. DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4-7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110,000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent Km values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl- were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by L-asparagine, nucleoside triphosphates except ATP, and sulfhydryl reagents. It has been observed that the properties of asparagine synthetase from rat liver are not so different from those of tumors such as Novikoff hepatoma and RADA 1.     

Regulation of mammalian asparagine synthetase by adenine nucleotides.

Initial velocity kinetic data indicate that ADP and AMP are inhibitors of mammalian liver asparagine synthetase. The non-product nucleotide ADP is a much more potent inhibitor than AMP, although both apparently compete for the same site. This modifier site, however, does not overlap spatially with the substrate site for ATP. Both ADP and AMP are V_max inhibitors, but ADP also raises the K_m for ATP. Adenylate energy charge, calculated at various levels of ATP and ADP show typical correlations with activity, but with AMP these correlations are weak and atypical.     

The nucleotide sequence of asparagine tRNA from Escherichia coli.

The nucleotide seuquence of Escherichia coli asparagine tRNA was determined to be pU-C-C-U-C-U-G-s4U-A-G-U-U-C-A-G-D-C-G-G-D-A-G-A-A-C-G-G-C-G-G-A-C-U-Q-U-U-t6A-A-phi-C-C-G-U-A-U-m G-U-C-A-C-U-G-G-T-phi-C-G-A-G-U-C-C-A-G-U-C-A-G-A-G-G-A-G-C-C-AOH. Its D-stem and D-loop have almost the same sequence as Escherichia coli aspartate tRNA.     

Aspartyladenylate analog--effective inhibitor of asparagine synthetase

A number of earlier unknown phosphonate analogues of aspartyl adenylate with anhydride oxygen substituted by --CH2--, and the carbonyl group substituted by --CH(OH)- or --CH(NH2)-groups were synthesized. These compounds were used to study the reaction mechanism of asparagine synthetases from white lupine and E. coli. The aspartyl adenylate analogues proved to be powerful competitive inhibitors (Ki = 10(-7) M) of the bacterial enzyme. In the case of white lupine enzyme catalyzing the aspartate-independent ATP--[32P]PPi exchange, the above compounds displayed a non-competitive type of inhibition with respect to aspartate and ATP, Ki = 10(-4) M. It is likely that for the latter enzyme the first intermediate is different from an aspartyl adenylate derivative.     

Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.