A comparative study of essential arginine residues in Gramicidin S synthetase 2 and isoleucyl tRNA synthetase

In gramicidin S synthetase 2 (GS 2) from Bacillus brevis, L-proline, L-valine, L-ornithine, and L-leucine activations to aminoacyl adenylates are progressively inhibited by phenylglyoxal. The inactivation of GS 2 obeys pseudo-first-order kinetics. ATP completely prevents inactivation of GS 2 by phenylglyoxal, whereas amino acids only partially prevent it. In the presence of ATP, four arginine residues per mol of GS 2 are protected from modification by phenylglyoxal as determined by amino acid analysis and the incorporation of [7-14C]phenylgloxal into the enzyme protein, indicating that a single arginine residue is necessary for each amino acid activation. In isoleucyl tRNA synthetase from Escherichia coli, phenylglyoxal inhibits activation of L-isoleucine to isoleucyl adenylate. ATP completely prevents inactivation, although isoleucine only partially prevents it. One arginine residue of isoleucyl tRNA synthetase is protected by ATP from modification by phenylglyoxal, suggesting that a single arginine residue is essential for isoleucine activation. These results support the involvement of arginine residues in ATP binding with GS 2 or isoleucyl tRNA synthetase, and thus indicate that arginine residues of amino acid activating enzymes are essential for the formation of aminoacyl adenylates in both nonribosomal and ribosomal peptide biosynthesis.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

Synthesis and activity of analogues of the isoleucyl tRNA synthetase inhibitor SB-203207

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.     

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.     

The nucleotide sequence for a proline-activating domain of gramicidin S synthetase 2 gene from Bacillus brevis

A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PPi exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoleucine auxotrophy as a consequence of a mutationally altered isoleucyl-transfer ribonucleic acid synthetase

Among mutants which require isoleucine, but not valine, for growth, we have found two distinguishable classes. One is defective in the biosynthetic enzyme threonine deaminase (l-threonine hydro-lyase, deaminating, EC 4.2.1.16) and the other has an altered isoleucyl transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5]. The mutation which affects ileS, the structural gene for isoleucyl-tRNA synthetase, is located between thr and pyrA at 0 min on the map of the Escherichia coli chromosome. This mutationally altered isoleucyl-tRNA synthetase has an apparent K(m) for isoleucine ( approximately 1 mm) 300-fold higher than that of the enzyme from wild type; on the other hand, the apparent V(max) is altered only slightly. When the mutationally altered ileS allele was introduced into a strain which overproduces isoleucine, the resulting strain could grow without addition of isoleucine. We conclude that the normal intracellular isoleucine level is not high enough to allow efficient charging to tRNA(Ile) by the mutant enzyme because of the K(m) defect. A consequence of the alteration in isoleucyl-tRNA synthetase was a fourfold derepression of the enzymes responsible for isoleucine biosynthesis. Thus, a functional isoleucyl-tRNA synthetase is needed for isoleucine to act as a regulator of its own biosynthesis.     

Characterization and location of the L-proline activating fragment from the multifunctional gramicidin S synthetase 2

Gramicidin S synthetase 2 (GS2) derived from Bacillus brevis is a multifunctional single polypeptide (Mr 280,000) with a 4'-phosphopantetheine residue covalently bound to the enzyme. When GS2 was treated with trypsin or chymotrypsin, fragments with some activity were liberated. The molecular mass of the L-proline activating fragment was 114 kDa on SDS-PAGE. This fragment, when incubated with gramicidin S synthetase 1 (GS1) in the presence of phenylalanine and proline, produced D-Phe-L-Pro dipeptide. The fragment accepted D-phenylalanine from GS1 in the absence of L-proline. The L-proline activating fragment was shown to lack pantothenic acid by microbiological assay. On the other hand, the L-leucine activating fragment, which was partially purified, contained a large amount of pantothenic acid, although it did not form the D-Phe-L-Pro dipeptide. These results indicate that the L-proline activating site is located near an acceptor site for D-phenylalanine on GS2, but that it is not adjacent to a 4'-phosphopantetheine group. The N-terminal sequence (15 amino acid residues) of the L-proline activating fragment obtained by trypsin treatment was identical with that of GS2, indicating that the L-proline activating site is located at the N-terminus of the native synthetase. The N-terminal sequence of GS2 has been matched with the amino acid sequence deduced from the nucleotide sequence 71 bp downstream of the stop codon of the GS1 gene except that the first initiator methionine was not detected.     

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Functional cysteinyl residues in human placental aldose reductase

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.     

Active site titration of gramicidin S synthetase 2: evidence for misactivation and editing in non-ribosomal peptide biosynthesis

The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PP(i) generation using active site titration measurements with [gamma-(32)P]ATP. The initial 'burst' of product formation can be correlated to the generation of the aminoacyl adenylate:enzyme complexes at the four amino acid activation domains and the subsequent aminoacylation of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amino acid substrates at a saturating concentration displayed a consumption of 8.3 ATP/GS2. In the presence of single amino acids, a binding stoichiometry higher than the anticipated two ATP per active site was obtained, implying misactivation at non-cognate domains. Breakdown of acyladenylate intermediates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation.     

Proofreading in trans by an aminoacyl-tRNA synthetase: a model for single site editing by isoleucyl-tRNA synthetase.

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code. Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine). That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme. Although IleRS.Val-AMP undergoes thiolysis as efficiently as do IleRS.Ile-AMP and IleRS.Ile-tRNAIle, IleRS.Val-tRNAIle does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS.Val-tRNAIle is, most likely, positioned off the enzyme.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Dependence of in vivo inactivation kinetics of gramicidin S synthetase on cell physiology

Gramicidin S synthetase, the enzyme complex catalyzing the biosynthesis of the antibiotic gramicidin S in Bacillus brevis, is subject to O(2)-dependent in vivo inactivation during exponential aerobic growth after reaching a peak in specific activity. The five amino acid substrates of the synthetase are capable of stabilizing its activity to varying degrees in whole cells shaken aerobically. Depending on the time of cell harvesting before, during, or after the peak in intracellular gramicidin S synthetase specific activity, the enzyme has a long, medium, or short half-life, respectively. The kinetic profiles of gramicidin S synthetase in B. brevis cells indicate that both the kinetics of synthetase loss and the degree of its amino-acid-mediated stabilization are a strong function of the cells' physiological development.     

A sulfhydryl presumed essential is not required for catalysis by an aminoacyl-tRNA synthetase

Several aminoacyl-tRNA synthetases are sensitive to reagents that modify sulfhydryl groups. We report here the significance of N-ethylmaleimide (NEM)-mediated inactivation of Escherichia coli glycyl-tRNA synthetase, and alpha 2 beta 2 enzyme. We confirmed earlier observations that NEM abolishes synthetase-catalyzed aminoacylation with pseudo-first order kinetics and provided a second method of proof that the site of inactivation is located in the beta-subunit. Using oligonucleotide-directed mutagenesis of the glyS gene, each beta-subunit cysteine codon (positions 98, 395, and 450) was replaced, individually, by an alanine codon. The three resulting mutant proteins are each active in vivo, and their in vitro aminoacylation activities are comparable to that of the native enzyme. A mutant incorporating all three amino acid substitutions is also active in vivo and in vitro. These results establish conclusively that a beta-subunit cysteine thiol is not required for the catalysis of aminoacylation. The Cys98----Ala and Cys450----Ala mutants are inactivated by NEM with the same kinetics as the wild-type protein. However, the Cys395----Ala mutant is refractory to NEM. This suggests that NEM inactivation of the native enzyme is due to alkylation of Cys395. Aware that inactivation may result from steric effects, we constructed a mutant with a bulkier amino acid residue at position 395 (Cys395----Gln). The aminoacylation activity of this protein is less than 10% of that of the wild-type enzyme. The glutamine substitution affects only the tRNA-dependent step of the reaction--the rate of glycyl adenylate synthesis is not lowered. In these features, the mutant resembles the NEM-inactivated protein. We propose that the NEM sensitivity of glycyl-tRNA synthetase, and possibly of other synthetases, arises from steric or conformational effects of the alkylated cysteine side chain.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

Sulfhydryl chemistry of Salmonella typhimurium phosphoribosylpyrophosphate synthetase: identification of two classes of cysteinyl residues

Sulfhydryl-specific reagents were used to study the reactivities and function of the four cysteinyl residues per subunit present in Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. In the presence of high concentrations of denaturants all four cysteinyl residues reacted with sulfhydryl-specific reagents. In the absence or in the presence of low levels of denaturing agents, two classes of cysteinyl residues were identified. A single sulfhydryl reacted rapidly with iodoacetamide and 5,5'-dithiobis(nitrobenzoic acid) (DTNB) without significant loss of enzymatic activity. This single sulfhydryl was identified as Cys-229 by reaction with iodo[1-14C]acetamide, followed by isolation and sequence analysis of a single radiolabeled peptide. The three remaining sulfhydryls reacted to various extents depending on the conditions and sulfhydryl-specific reagents employed. At low Pi concentrations, these residues reacted fully with DTNB, leading to an 80 to 90% loss of enzymatic activity. ATP and high levels of Pi prevented this reaction. These results, along with studies comparing the S. typhimurium PRPP synthetase sequence with the sequences of PRPP synthetases from other species, suggest that the cysteinyl residues in the Salmonella enzyme are not catalytically essential. That one or more of the three less reactive residues may lie in or near the active site is not excluded.     

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by oxidation of a specific sulfhydryl group with potassium permanganate.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.     

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.