Structural analyses of a human lysyl-tRNA synthetase mutant associated with autosomal recessive nonsyndromic hearing impairment.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Procedure for Purification of Recombinant preMsk1p from E. coli Determines Its Properties as a Factor of tRNA Import into Yeast Mitochondria.

Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E. coli in the denatured state. We also discuss the possible mechanistic reasons for this phenomenon.

Solid-phase combinatorial synthesis of a lysyl-tRNA synthetase (LysRS) inhibitory library.

The solid-phase combinatorial synthesis of a new library with potential inhibitory activity against the cytoplasmic lysyl-tRNA synthetase (LysRS) isoform of Trypanosoma brucei is described. The library has been specifically designed to mimic the lysyl adenylate complex. The design was carried out by dividing the complex into four modular parts. Proline derivatives (cis-gamma-amino-L-proline or trans-gamma-hydroxy-L-proline) were chosen as central scaffolds. After primary screening, three compounds of the library caused in vitro inhibition of the tRNA aminoacylation reaction in the low micromolar range.

Multiple forms of arginyl- and lysyl-tRNA synthetases in rat liver: a re-evaluation.

The size distribution of lysyl- and arginyl-tRNA synthetases in crude extracts from rat liver was re-examined by gel filtration. It is shown that irrespective of the addition or not of several proteinase inhibitors, lysyl-tRNA synthetase was present exclusively as a high-Mr entity, while arginyl-tRNA synthetase occurred as high- and low-Mr forms, in the constant proportions of 2:1, respectively. The polypeptide molecular weights of the arginyl-tRNA synthetase in these two forms were 74000 and 60000, respectively. The high-Mr forms of lysyl- and arginyl-tRNA synthetases were co-purified to yield a multienzyme complex, the polypeptide composition of which was virtually identical to that of the complexes from rabbit liver and from cultured Chinese hamster ovary cells. Of the nine aminoacyl-tRNA synthetases, specific for lysine, arginine, methionine, leucine, isoleucine, glutamine, glutamic and aspartic acids and proline, which characterize the purified complex, each, except prolyl-tRNA synthetase, was assigned to the constituent polypeptides by the protein-blotting procedure, using the previously characterized antibodies to the aminoacyl-tRNA synthetase components of the corresponding complex from sheep liver.

Multiple molecular forms of cysteinyl-tRNA synthetase from rat liver: purification and subunit structure.

Cysteinyl-tRNA synthetase (L-cysteine:tRNACys ligase (AMP-forming), EC 6.1.1.16) has been purified from rat liver in 23% overall yield. The enzyme was resolved by hydroxyapatite chromatography into three active forms (Fractions CRS-1, CRS-2 and CRS-3). The total activity ratio was about 0.7:2:1. The fractions CRS-2 and CRS-3 contained no other detectable aminoacyl-tRNA synthetase activity. CRS-2 was homogeneous by polyacrylamide gel electrophoresis, CRS-3 gave two active bands with mobilities corresponding to those of CRS-1 and CRS-2. The molecular weight of CRS-2 was about 240 000 by electrophoretic mobilities on the gels of various porosity, and 115 000-140 000 by sucrose gradient centrifugation. By gel-filtration, CRS-1, CRS-2 and CRS-3 exhibited apparent molecular weights of 122 000, 235 000 and 300 000, respectively. By sodium dodecyl sulfate gel electrophoresis, both CRS-2 and CRS-3 gave a single major band of 120 000 daltons. Stoichiometric study of cysteinyl adenylate formation indicated that CRS-2 has two active sites per molecule. These results are consistent with a dimeric structure of the type alpha2 for the major form of rat liver cysteinyl-tRNA synthetase, composed of two probably identical subunits of about 120 000 daltons. Available evidence also suggests that CRS-1 and CRS-3 are alpha and alpha3 (or alpha4), respectively.

Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsPro and tRNAsLys by homologous and heterologous enzymes.

The cytoplasmic prolyl-tRNA synthetase can be separated by hydroxyapatite chromatography, from the enzyme present in the chloroplasts and in the mitochondria (organellar enzyme). The cytoplasmic lysyl-tRNA synthetase can also be separated from the organellar enzyme. There are two tRNAsPro in the cytoplasm; they can be charged by the cytoplasmic enzyme, but not by the organellar enzyme or the Escherichia coli enzyme. Chloroplasts contain, in addition to the two cytoplasmic tRNAsPro, one chloroplast-specific tRNAPro, which is not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. Mitochondria contain, in addition to the two cytoplasmic tRNAsPro, two mitochondria-specific tRNAsPro, which are not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. There are two tRNAsLys in the cytoplasm. Both can be charged by the cytoplasmic enzyme, but one can be charged by the organellar or E. coli enzyme. Chloroplasts contain in addition to one cytoplasmic tRNALys, one chloroplast-specific tRNALys which can only be charged by the organellar or E. coli enzyme. Mitochondria contain, in addition to one cytoplasmic tRNALys, one mitochondria-specific tRNALys which can only be charged by the organellar or E. coli enzyme.

Overproduction and purification of lysyl-tRNA synthetase encoded by the herC gene of E coli.

Lysyl-tRNA synthetases are synthesized from two distinct genes in E coli, lysS and lysU, but neither gene product has been purified distinctively by using overproducing systems. The lysS gene has been identified by a herC mutation which restores maintenance of the mutant ColE1 replicon. The herC gene product was overproduced by using a tac promoter fusion and purified to homogeneity. The purified HerC protein possesses a lysyl-tRNA synthetase activity as predicted by the sequence identity of herC to lysS. The procedure is useful for rapid mass-scale purification of lysyl-tRNA synthetase.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Purification, and fluorometric and kinetic analysis of the binding of substrates, L-lysine and ATP.

Lysyl-tRNA synthetase [L-lysine:tRNA(Lys)ligase (AMP forming); EC 6.1.1.6] was purified from Bacillus stearothermophilus NCA1503 approximately 1,100-fold to homogeneity in PAGE. The enzyme is a homodimer of M(r) 57,700 x 2. The molar absorption coefficient, epsilon, at 280 nm is 71,600 M-1.cm-1 at pH8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PPi exchange reaction increases up to 50 degrees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optima of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 muM, respectively, and in the ATP-PPi exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 muM, respectively at 37 degrees C, pH 8.0. Interaction of either L-lysine or ATP with the enzyme has been investigated by using as a probe the ligand-induced quenching of protein fluorescence and by equilibrium dialysis. These static analyses, as well as the kinetic analysis of the L-lysine dependent ATP-PPi exchange reaction indicate that the binding mode of L-lysine and ATP to the enzyme is sequential ordered (L-lysine first). The interaction of lysine analogues with the enzyme has also been investigated.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Formation and isolation of an enzyme-lysyladenylate complex and its analogue.

The formation of an enzyme.lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNALYS ligase (AMP-forming); EC 6.1.1.6] from Bacillus stearothermophilus. The apparent dissociation equilibrium constants of the enzyme with L-lysine and ATP in the process of the complex formation were estimated to be 50.9 and 15.5 microM, respectively, at pH 8.0, 30 degrees C, by fluorometric measurement. The isolated enzyme.lysyladenylate complex was relatively stable with a rate constant of decomposition of 1.7 x 10(-5) s-1 at pH 8.5 and 0 degree C. The rate constant of transfer of L-lysine from the complex to Escherichia coli tRNA was 1.2 x 10(-2) S-1 at pH 8.5 and 0 degree C. The effects of replacing L-lysine by several analogues on the complex formation were examined. L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PPi exchange reaction, produced a stable complex with the enzyme and ATP, enzyme.lysinehydroxamate-AMP probably being formed. The binding stoichiometry of the assumed L-lysinehydroxamate-AMP per mol of the dimer enzyme was 1:1.

[Isolation and properties of lysyl-tRNA-synthetase from rabbit skeletal muscles]. / Vydelenie i svoistva lizil-tRNK-sintetazy iz skeletnykh myshts krolika.

A method is developed to isolate lysyl-tRNA-synthetase from 93-95%-purity postribosomal supernatant fraction of skeletal muscle homogenate in rabbit. Novelty of the method is the ATP usage for muscle homogenization, which permits shortening the number of operations during the enzyme isolation. The molecular weight of protein is 68 +/- 10 kDa, the monomer unit consists of 540 amino acids and contains a carbohydrate component.

[A new method of isolating lysyl-tRNA-synthetase from the rat liver]. / Novyi metodicheskii podkhod k vydeleniiu lizil-tRNK-sintetazy iz pecheni krys.

The methods of gel filtration on sepharose 6B, hydrophobic chromatography, chromatofocusing were used to isolate the preparation of lysyl-tRNA-synthetase. The enzyme activity at the terminal stage of purification is 1800 times as high. The isolated preparation is homogeneous with electrophoresis in 4-30% PAAG.

[Physico-chemical properties of lysyl-tRNA-synthetase from the rat liver]. / Fiziko-khimicheskie svoistva lizil-tRNK-sintetazy pecheni krys.

Two lysyl-tRNA-synthetase forms are obtained from the rat liver. Their molecular masses are determined by electrophoresis and gel-filtration on Sephadex G-150: form I-122, form II-64 kDalton. Gel-electrophoresis in the presence of 0.1% SDS indicates that form I of lysyl-tRNA-synthetase consists of two subunits with a molecular mass of 64 kDalton each, i. e. it is a dimer. Optimal conditions and kinetic parameters (Km and Vmax) of aminoacylation for the both enzyme forms are similar. Amino acid composition, fluorescence parameters and thermal inactivation conditions are determined.

Functional association between three archaeal aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA(Pro) aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K(D) of about 0.3-0.9 microm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNA(Lys) over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.

Multiple forms of lysyl-tRNA synthetase from Escherichia coli.

Lysyl-tRNA synthetase has been isolated from E. coli. The enzymatic activity elutes as three or four bands from a hydroxyl-apatite column. Polyacrylamide gel analysis shows that each of these bands contain more than one enzymatically active protein species. The molecular weights of the subunits of these species provides an explanation for the variation in the molecular weights previously reported for this enzyme.

Purification and characterization of lysyl-tRNA synthetase after dissociation of the particulate aminoacyl-tRNA synthetases from rat liver.

The major aminoacyl-tRNA synthetase complex (the 24 S complex) isolated from rat liver, which contains lysyl-, leucyl-, and arginyl-tRNA synthetase activities, is dissociated into fully active free aminoacyl-tRNA synthetases by column chromatography on diaminooctyl-Sepharose. During the hydrophobic interaction chromatography, more than a quantitative yield of the lysyl-tRNA synthetase activity is obtained. The free lysyl-tRNA synthetase, dissociated from the synthetase complex, is purified about 2,000-fold with a 13% yield by conventional column chromatography. Lysyl-tRNA synthetase is also purified from the 24 S synthetase complex by affinity column chromatography on lysyl-diaminohexyl-Sepharose. Free lysyl-tRNA synthetase as dissociated from the synthetase complex, is evidently a dimeric enzyme with a subunit molecular weight of 66,000 +/- 3,000, as determined by gel electrophoresis, sucrose gradient centrifugation and gel filtration.

Rapid isolation of lysyl-tRNA synthetase from rat liver.

The high molecular weight aminoacyl-tRNA synthetase complex (the 24S complex) was isolated from rat liver by ultracentrifugation. The lysyl-tRNA synthetase (E.C. 6.1.1.6) was selectively dissociated by hydrophobic interaction chromatography on 1,6 diaminohexyl agarose followed by hydroxylapatite chromatography and DEAE chromatography. The lysyl-tRNA synthetase dissociated from the 24S synthetase complex was purified approximately to 2700 fold with 14% yield.

Purification and kinetic mechanism of lysyl-tRNA synthetase from Mycobacterium smegmatis SN2.

Lysyl-tRNA synthetase [L-lys:tRNAlys ligase (AMP forming) EC:6.1.1.6] has been purified to homogeneity from Mycobacterium smegmatis SN2. The enzyme is a dimer of molecular weight 126,000 and is composed of identical subunits. A detailed analysis of the kinetic mechanism of the lysyl-tRNA synthetase has been carried out. A rapid equilibrium random ter ter mechanism is proposed based on initial velocity and product inhibition studies. There is no evidence for the formation of enzyme-bound lysyl-adenylate. The reverse reaction, studied by the deacylation of lysyl-tRNA, requires the presence of both AMP and PPi. This observation is consistent with the mechanism proposed.

Lysyl-tRNA synthetase.

Lysyl-tRNA synthetase catalyses the formation of lysyl-transfer RNA, Lys-tRNA(Lys), which then is ready to insert lysine into proteins. Lysine is important for proteins since it is one of only two proteinogenic amino acids carrying an alkaline functional group. Seven genes of lysyl-tRNA synthetases have been localized in five organisms, and the nucleotide and the amino acid sequences have been established. The lysyl-tRNA synthetase molecules are of average chain lengths among the aminoacyl-tRNA synthetases, which range from about 300 to 1100 amino acids. Lysyl-tRNA synthetases act as dimers; in eukaryotes they can be localized in multienzyme complexes and can contain carbohydrates or lipids. Lysine tRNA is recognized by lysyl-tRNA synthetase via standard identity elements, namely anticodon region and acceptor stem. The aminoacylation follows the standard two-step mechanism. However the accuracy of selecting lysine against the other amino acids is less than average. The first threedimensional structure of a lysyl-tRNA synthetase worked out very recently, using the enzyme from the Escherichia coli lysU gene which binds one molecule of lysine, is similar to those of other class II synthetases. However, none of the reaction steps catalyzed by the enzyme is clarified to atomic resolution. Thus surprising findings might be possible. Lysyl-tRNA synthetase and its precursors as well as its substrates and products are targets and starting points of many regulation circuits, e.g. in multienzyme complex formation and function, dinucleoside polyphosphate synthesis, heat shock regulation, activation or deactivation by phosphorylation/dephosphorylation, inhibition by amino acid analogs, and generation of antibodies against lysyl-tRNA synthetase. None of these pathways is clarified completely.

Physical and biochemical characterization of a purified arginyl-tRNA synthetase-lysyl-tRNA-synthetase complex from rat liver.

Arginyl- and lysyl-tRNA synthetases copurify throughout a six-step chromatographic procedure resulting in a purification of 605- and 559-fold, respectively. The purified enzymes were estimated to be 98% pure with a stoichiometry of 1:1 from acrylamide gel electrophoresis under denaturing conditions. On the basis of a native molecular weight of 285000 calculated from s20,w, Rs, and V and subunit molecular weights of 73000 and 65000 obtained by sodium dodecyl sulfate gel electrophoresis, the synthetases appear to exist as a tetramer. The tetrameric structure was also supported by cross-linking studies. These results are consistent with an alpha 2 beta 2 structure, but an alpha beta structure has not been ruled out.

Comparison of complexes containing lysyl-tRNA synthetase from normal and virus-transformed cells.

We compared the lysyl-tRNA synthetases from normal (Balb/3T3) and murine sarcoma virus-transformed (KA31) mouse fibroblasts. In agreement with several other reports of mammalian systems, the lysyl-tRNA synthetases from these cells occurred in very large postmicrosomal complexes as determined by gel filtration on agarose columns. Arginyl-, isoleucyl-, methionyl-, phenylalanyl-, and tyrosyl-tRNA synthetases also occurred as part of a large complex or complexes. Activity of glycyl- or leucyl-tRNA synthetase was not detected in a complex. The specific activities of arginyl- and methionyl-tRNA synthetases were three- and five-fold higher, respectively, in a complex from KA31 as compared with a complex from Balb/3T3. In contrast, the specific activity of lysyl-tRNA synthetase from the Balb/3T3 complex was 50% higher than that of the KA31 complex. tRNALys obtained from the complexes of Balb/3T3 and KA31 was fractionated into isoacceptors on columns of RPC-5. The relative amounts of lysine isoacceptors in total preparations of tRNA from normal whole cells and in tRNA obtained from the normal enzyme complex were the same. However, two isoacceptors were present in greater amounts and two were present in lesser amounts in the KA31 enzyme complex as compared with lysine isoacceptors in a total preparation of tRNA from KA31 cells.