Search

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNA in a two-step reaction starting with amino acid activation followed by aminoacyl group transfer to tRNA. To clear mistakes that occasionally occur, some of these enzymes carry out editing activities, acting on the misactivated amino acid (pretransfer editing) or after the transfer on the tRNA (post-transfer editing). The post-transfer editing pathway of leucyl-tRNA synthetase has been extensively studied by structural and biochemical approaches. Here, we report the finding of a tRNA-independent pretransfer editing pathway in leucyl-tRNA synthetases from Aquifex aeolicus. Using a CP1-mutant defective in its post-transfer editing function, we showed that this new editing pathway is distinct from the post-transfer editing site and may occur at the synthetic catalytic site, as recently proposed for other aminoacyl-tRNA synthetases.

In human cytoplasm, nine aminoacyl-tRNA synthetases (aaRSs) and three protein factors form a multi-synthetase complex (MSC). Human cytosolic methionyl-tRNA synthetase (hcMetRS) is a component of the MSC. Sequence alignment revealed that hcMetRS has an N-terminal extension of 267 amino acid residues. This extension can be divided into three sub-domains: GST-like, GN, and GC sub-domains. The effect of each sub-domain in the N-terminal extension of hcMetRS on enzymatic activity and incorporation into the MSC was studied. The results of cellular assay showed that the GST-like sub-domain was responsible for the incorporation of hcMetRS into the MSC. The entire N-terminal extension of hcMetRS is indispensable for the enzymatic activity. Deletion mutagenesis revealed that a seven-amino acid motif within the sub-domain GC was important for the activity of amino acid activation. A conserved proline residue within the seven-amino acid motif was crucial, while the other six residues were moderately important for the amino acid activation activity. Thus, the last 15 residues of previously defined N-terminal extension of hcMetRS was a part of the catalytic domain; whereas the first 252 residues of hcMetRS constitute the N-terminal extended domain of hcMetRS. The formerly defined N-terminal extension of hcMetRS possesses two functions of two different domains.

Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.

Leucyl-tRNA synthetase (LeuRS) contains an editing domain that discriminates leucine from noncognate amino acids to ensure translational fidelity. In this study, a knock-out strain for Saccharomyces cerevisiae LeuRS was constructed to analyze in vivo the tRNA aminoacylation properties of S. cerevisiae and human cytoplasmic LeuRSs. The activities of several editing-defective mutants of ycLeuRS were determined in vitro and compared with those obtained in vivo in a complementation assay performed in the knock-out strain. The editing activities of these mutants were analyzed in the presence of either norvaline, a leucine analogue, or AN2690, a specific inhibitor that targets the editing active site. In general, the in vivo data are consistent with those obtained in vitro. Our results show that ycLeuRS post-transfer editing plays a crucial role in the establishment of the aminoacylation fidelity. When impaired, the viability of cells bearing editing-defective mutants is drastically decreased in the presence of noncognate amino acid. This study also emphasizes the crucial function of some semi-conserved residues around the editing site in modulating the editing efficiency. The assay system can be used to test the effect of compounds that potentially target the aminoacylation or editing active site of fungal LeuRS.

Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. The non-catalytic domains usually have distinct roles in the aminoacylation reaction. Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) is composed of a separated catalytic site and tRNA anticodon-binding site, which would represent one of the closest relics of the primordial aminoacyl-tRNA synthetase. Moreover, the essential catalytic site residues are split into the two different subunits. In all other class-I aminoacyl-tRNA synthetases, those two functional polypeptides are nowadays fused into a single protein chain. In this work, we report the isolation and the characterization, in Escherichia coli, of a novel oligomeric form (alphabeta)2 for A. aeolicus LeuRS, which is present in addition to the alphabeta heterodimer. A. aeolicus (alphabeta)2 LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alphabeta)2 enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer alphabeta can bind one tRNALeu molecule whereas the heterotetramer (alphabeta)2 can bind two tRNALeu molecules. Active site titration and aminoacylation assays showed that two functional active sites are found per heterotetramer, suggesting that this molecular species might exist and be active in vivo. All those data suggest that the existence of the heterotetramer is certainly not an artifact of overexpression in E. coli.

The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 A. Crystals diffract to beyond 1.8 A resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 A resolution. All structures were solved by molecular-replacement techniques.

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.

The Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the beta subunit which is believed to bind tRNA(Leu). A yeast three-hybrid system was constructed on the basis of the interaction of the beta subunit with its cognate tRNA(Leu). Then, seven mutated beta subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated beta subunits and on the co-expressed alphabeta-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.

Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to class I aminoacyl-tRNA synthetases. It consists of 860 amino acid residues and catalyzes the leucylation of tRNA(Leu). An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C. Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His(6)-tag at its N-terminus was constructed to facilitate the purification of His(6)-LeuRS-C through one-step affinity chromatography on Ni(2+)-NTA column. The purified His(6)-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His(6)-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His(6)-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His(6)-LeuRS, respectively.

Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292-A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the Km values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.

Glutaryl 7-amino cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp.130 catalyzes hydrolysis of glutaryl 7-amino cephalosporanic acid to produce 7-amino cephalosporanic acid (7-ACA). 7-ACA is the starting material for the industrial production of most cephalosparonic derivatives. Six plasmids for expression of GL-7ACA acylase were constructed and these recombin ant plasmids presented different expression characteristics in Escherichia coli. The acylase gene from plasmid pKKCA1 was inserted into plasmid pMFT7-5 and the resulting plasmid pMFT7CA1 has higher expression in E.coli. The specific activity of the crude extract of the transformant JM109(DE3)/pMFT7CA1 was near 5 u/g, so the over produced enzyme was easily purified by a single-step anion exchange column chromatography. The enzyme could be purified by immobilized ion affinity chromatography after fused by 6xHis in the N-terminal of its alpha-subunit. Because plasmid pSMLCA1 brings tc(R) and p15A origin, it is special useful plasmid in fermentation. Two secretory expression plasmids, pSUCA1S and pETCA1pelB, could secrete the acylase to periplasmic space of bacteria. The whole cells containing the secretory expression plasmid may be used for production of 7-ACA directly.

An Asn(2) deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn(2) and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied. The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in aminoacid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNA(Arg) at all. The mutant deleted Asn(2) and Ile(3) was able to be expressed in Escherichia coli but not stable to be purified. The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme. The data show that the conformation of the mutants are different and the tryptophan residues in the mutants are more exposed than those in the native enzyme. An estimate of the secondary structure of the mutant enzymes from their far ultraviolet CD spectra showed that the chimera mutant contained less alpha-helix, more beta-sheet and slightly higher fraction of random coil, as compared with the native enzyme. The results indicate that an intact N-terminal domain of E.coli arginyl-tRNA synthetase is important to its activity and correct folding.

The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp. 130 were determined. The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp. GK16 and C427, but low homology with the others. There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation.

Previous studies showed that the gene argS encoding the arginyl-tRNA synthetase(ArgRS) from Escherichia coli(E.coli), was overexpressed 1 000 folds in the E.coli transformant TG1/pUC-argS, while the gene leuS, encoding the leucyl-tRNA synthetase(LeuRS) from E.coli, was only overproduced 35-fold in the same case. To investigate why the expression of these two aminoacyl-tRNA synthetase genes is so different, a fused gene (termed parg-leuS) was constructed by replacement of the 5' flanking region of leuS to 5' flanking region of argS. In the E.coli transformant TG1/pUC-parg-leuS, the activity of LeuRS was only improved 8.5-fold, which was much lower than that of the transformant harboring the recombinant plasmid pUC18-leuS or pKK-leuS. However, by RNA dot hybridization the amount of mRNA produced in the transcription of parg-leuS was about 5 times than that of the wild type leuS, and was similar to that of pKK-leuS, suggesting that the promoter of argS is very strong. Analysis of the secondary structure around the initiation codon among three mRNAs showed that the secondary structure of the mRNA from parg-leuS was the strongest of the three mRNAs. From the results, it could be deduced that expression of the fused gene parg-leuS might be controlled at the translational level and the strong secondary structure of this mRNA may hinder translation initiation and result in a low translation efficiency.

The gene argS encoding arginyl-tRNA synthetase (ArgRS) in Escherichia coli has been cloned into the vector pMFT7-5 which allows overproduction of ArgRS. The specific activity of the synthetase in the crude extract of E.coli JM109(DE3) transformant containing the plasmid pMFT7-argS was approximately 2 500 times that of JM109(DE3) (the host strain without the vector). The overproduced synthetase can be purified to homogeneity with the specific activity of 36 000 units/mg under native condition within one day by a two-step purification system of DEAE-Sepharose CL-6B Fast Flow and Blue Sepharose CL-6B chromatographies with an overall yield of 69%. Moreover, this system makes it very easy to incorporate efficiently the expensive labeled amino acids into this enzyme. This novel system can conveniently provide a large amount of purified enzyme with a much higher specific activity than before which may be beneficial to NMR and crystallographic studies on this enzyme.

A synthetic gene encoding Escherichia coli tRNA(Arg)(2) was inserted in a plasmid under the control of an isopropyl-beta, D-thiogalactopyranoside (IPTG)-inducible promotor, pTrc99B. In E.coli MT102 transformed by the above plasmid containing the target gene. TRNA(Arg)(2) was overproduced up to 30 fold of that of the host. In the transformant the quantity contained tRNA(Arg) increased 10 times and was 70% of the total tRNA. The tRNA(Arg)(2) was purified to 88% homogeneity by passing through a DEAE-Sephacel column, and then was purified by benzyl-DEAE cellulose column chromatography to a purity of 99% with an arginylation activity of 1 600 pmole/A(260) unit. Eighteen milligrams of tRNA(Arg)(2) could be obtained from 40 mg total tRNA which was obtained from four liters of overnight culture, and the yield of the purification was 62%. The accurate kinetic constants of aminoacylation of tRNA(Arg)(2) catalyzed by arginyl-tRNA synthetase were comparable with that of tRNA(Arg) from Sigma.

Site-directed mutagenesis and chemical modification were performed at Ser290 of the penicillin G acylase from E. coli ATCC11105. The Ser290 was substituted by Cys or Secys. Wild type and mutant proteins were purified, and the activities and kinetic constants of penicillin acylases for hydrolysis and synthesis were determined, respectively. Although their K(m) values were not changed, the k(cat) values of the thiol-PGA and seleno-PGA were decreased from 135s(-1) to 0.63s(-1) and 0.38s(-1) against NIPAB, and from 34.38s(-1) to 0.23s(-1) and 0.06s(-1) against penicillin G. Contrary to Choi's report(Choi K S (et al. J Bacteriology), 1992, 10 6270-6276), we found that hydrolysis activity was certainly kept in the mutant of penicillin acylase. In addition, the specific activities of synthesis were decreased by 5-fold and 20-fold, respectively.

In order to study structure and function of E.coli arginyl-tRNA synthetase(ArgRS), deletion of R245 and 252 in this enzyme was carried out by site-directed mutagenesis of gene coding for this enzyme, respectively. The genes, argSdeltar245 and argSdeltar252-encoding ArgRS mutant, ArgRSdeltaR245 and ArgRSdeltaR252 were obtained and cloned into pUC18 or pTrc99B, respectively. The expression of argSdeltar245 and the properties of the mutant ArgRSdeltaR245 were studied. The gene encoding ArgRS, argS was expressed as a soluble protein; however, ArgRS deleted R245 formed inclusion body during the expression of argSdeltar245 in E. coli. After dissolution of the inclusion body by 8 mol/L urea or 7 mol/L guandidine chloride, followed by renaturation by the methods of gel filtration or dilution, the specific activity of the mutant enzyme, ArgRSdeltaR245 was about 40 unit/mg, that is only 0.3% activity of the native enzyme. By detection of ultraviolet absorbance and difference spectroscopy and titration of DTNB, the conformations of the native and mutant enzymes were compared. It was shown that the tertiary structure of ArgRSdeltaR245 was more relaxed than that of ArgRS, and the chromophoric groups in the mutant enzyme were more exposed than that of the native enzyme. It may be the main reason of forming inclusion body during the mutant gene expression and losing activity of enzyme.

The gene of beta-subunit of GL-7-ACA acylase [7beta-(4-carboxybutanamido) cephalosporanic acid acylase] was cloned into pTrc99B, an IPTG inducible pasmid, to form the recombinant called pTrc-CA1B. Another recombinant plasmid pTrcCA1S was obtained by cloning the gene encoding alpha-subunit of GL-7-ACA acylase, the signal peptide and the expression elements from Pseudomonas sp. into pTrcCA1B. Then, recombinant plasmid pKKCA1S was constructed by cloning the gene encoding the signal peptide, expression elements and GL-7-ACA acylase into the vector pKK235. pTrcCA1S and pKKCA1S were allowed to transform TG1. These two plamids were able to transfer the expression product into the periplasmic space of the host bacteria. As a result, in the whole cell of TG1/pTrcCAIS, the specific activity of GL-7-ACA acylase was 23.9 u/g cell, 8 fold higher than that of TG1/pMR24. And in the whole cell of TG1/pKKCA1S, the specific activity of acylase was 18.3 u/g cell, 6 fold higher than that of TG1/pMR24.

Arginyl-tRNA synthetase (ArgRS) from E. coli was overproduced from transformant containing the gene encoding this enzyme (argS) by 550 fold higher than that from host cell. By site-directed mutagenesis the cleavage site for NcoI restriction endonuclease was introduced into the start codon of argS. The mutant gene was recombinated with plasmid pTrc99B under IPTG control. In the transformant containing the recombinated plasmid, argS can overexpressed 2 000 times higher than that in host cell. Through one step DEAE-Sepharose column chromatography, ArgRS was purified to be one band by SDS-PAGE and its specific activity was 15 000 u/mg, similar to reported values. The mutant ArgRS2ND which had the second residue asparagine replaced by aspartic acid showed no change of activity, kinetic constants nor the thermal and denaturational stabilities.

The 3.2 kb gene (leuS) encoding for leucyl-tRNA synthetase (LeuRS) has been cloned from E.coli K-12, and overexpressed 35 times more than that in the host strain TG1. In order to further increase the production of LeuRS, two types of leuS with different length of the 3' flanking region: leuS1 has an additional 130 bp over that of leuS2 were ligated into pKK-233-2 and pTrc-99B, respectively. In E. coli TG1 transformant harboring the recombination plasmid pTrc-99B with leuS2, the yield of LeuRS was 135 times that in TG1 strain. The purified enzyme that showed one band on SDS-PAGE was obtained after two steps of column chromatography. During the construction of plasmid, a substitute of G for C was introduced at position of base 4 in the coding region of leuS, so that Gln2 of LeuRS was changed to Glu. This enzyme was designated LeuRS2E. The kinetic constants of LeuRS2E showed that the substitution has no effect on the enzyme activity and could be used in the studies of LeuRS as the native enzyme.

The conformation of native enzyme ArgRS and its mutants ArgRS306KA, ArgRS306KR and ArgRS381KA was studied by absorbance spectrum, Difference spectrum with solvent perturbation, fluorescence spectrum and CD spectrum. The results showed that the chromophores of ArgRS306KR and ArgRS306KA had shifted to different micro environments in comparison to the native enzyme. Compared to the native enzyme, ArgRS306KA had and even bigger conformational change than ArgRS306KR, while the conformation difference between ArgRS381KA and ArgRS was not big enough to be perceivable in the spectrum. The analysis of CD spectrum showed the less the percentage of beta-turn in the ArgRS mutants, the lower the activity of the mutants. The conclusion can be drawn that the positive charge of Lys306 is very important to maintain the conformation of ArgRS, the change of conformation might be mainly responsible for the loss of enzyme of the mutants, while the substitution of Lys381 seems to cause no perceivable change of the enzyme conformation.