An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures.

Urzymes-short, active core modules derived from enzyme superfamilies-prepared from the two aminoacyl-tRNA synthetase (aaRS) classes contain only the modules shared by all related family members. They have been described as models for ancestral forms. Understanding them currently depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS's, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of (15)N tryptophanyl-tRNA synthetase Urzyme by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of (1)H chemical shifts (0.5 ppm) is inconsistent with a narrow ensemble of well-packed structures in either free or substrate-bound forms, although the number of resonances from the bound state increases, indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of helices and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorescence at ambient temperatures. Although the term "molten globule" is difficult to define precisely, these characteristics are consistent with most such definitions. Active-site titration shows that a majority of molecules retain ∼60% of the transition state stabilization free energy observed in modern synthetases. In contrast to the conventional view that enzymes require stable tertiary structures, we conclude that a highly flexible ground-state ensemble can nevertheless bind tightly to the transition state for amino acid activation.

Fractionation of plant aminoacyl-tRNA synthetases on tRNA-Sepharose columns.

It has been shown that tRNA-Sepharose, a chromatographic adsorbent containing unfractionated tRNA bound to a Sepharose matrix, is a useful, group-specific adsorbent for fractionation of the plant aminoacyl-tRNA synthetases. Conditions are described in which Val-, Trp-, Phe-, Leu- and Ile-tRNA synthetases from yellow lupin seeds can be separated from each other on the tRNA-Sepharose columns. Factors affecting affinity chromatography on the t-RNA-Sepharose columns are discussed. The affinity chromatography procedure for the purification of lupin Ser-tRNA synthetase to homogenity is described.

A concerted tryptophanyl-adenylate-dependent conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase revealed by the fluorescence of Trp92.

A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNATrp binding and recognition. The crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy. B. subtilis TrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNATrp is unlikely. Time-resolved fluorescence indicates conformational heterogeneity of B. subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed-TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure. The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an alpha-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 angstroms) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local alpha-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small alpha-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.

Carbohydrates in mammalian tryptophanyl-tRNA synthetase.

Homogeneous preparations of bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) contain monosaccharides (mannose, fucose, galactose, N-acetylglucosamine) as revealed by liquid chromatography. Their content comprises 2.5-3.0% (w/w) of the enzyme composed of two subunits (60 kDa x 2). The same set of sugars was detected in elastase and CNBr-generated fragments (with molecular masses of approx. 40 kDa and 30 kDa, respectively). It is concluded that bovine tryptophanyl-tRNA synthetase, in addition to being a metallo- and phosphoprotein, is also a glycoprotein.

Ap3A and Ap4A are primers for oligoadenylate synthesis catalyzed by interferon-inducible 2-5A synthetase.

The biological role of Ap3A synthesized in cells by tryptophanyl-tRNA synthetase (WRS) is unknown. Previously we have demonstrated that the cellular level of Ap3A significantly increases after interferon treatment. Here we show that the human 46 kDa 2-5A synthetase efficiently utilizes Ap3A as a primer for oligoadenylate synthesis. The Km for Ap3A is several-fold lower than for Ap4A and 100-fold lower than for ATP. This implies that Ap3A might be a natural primer for the 2'-adenylation reaction catalysed by 2-5A synthetase. Since WRS and 2-5A synthetase are both interferon-inducible proteins, a new link between two interferon-dependent enzymes is established.

[Tryptophanyl tRNA synthetase: isolation and characteristics of the tryptophanyl-enzyme]. / Triptofanil-tRNK-sintetaza: vydelenie i kharakteristika triptofanil-fermenta.

The catalytic groups, involved in aminoacyl-tRNA formation remain unknown. The isolation and identification of an active covalent complex between the enzyme and substrate is an essential step in understanding the reaction mechanism. We identified and isolated the covalent complex of tryptophanyl-tRNA synthetase (EC 6.1.1.2) and tryptophane which was able to aminoacylate the tRNATrp in the absence of ATP. In beef pancreas tryptophanyl-tRNA synthetase preparations, isolated by the previously described method, a tightly bound tryptophan was revealed which could not be removed by charcoal treatment, by gel-filtration and by replacement with the excess of typtamine, a competitive inhibitor of tryptophane. This tightly bound tryptophane is able to exchange rapidly and specifically with radioactive tryptophane allowing to obtain [14C]tryptophane-tryptophanyl-tRNA synthetase complex. After the reaction of this complex with NH2OH at neutral pH tryptophanyl hydroxamate is formed proving the activated state of the tryptophane in the initial complex with the enzyme. No nucleotide impurites were noticed in the enzyme preparation; the complex is stable at denaturation. A conclusion is made that the tryptophanyl-tRNA synthetase isolated by our method is a tryptophanyl-enzyme. The tryptophanyl residue could be specifically transferred to tRNATrp in the absence of other substrates of the reaction, the efficiency of the transfer does not exceed 25%. The content of the covalently bound tryptophane never exceeds 1 mole per mole of the dimeric enzyme. The total content of tryptophane in the forms of tryptophanyl-enzyme and tryptophanyl adenylate enzyme complex equals 2 moles per mole of the enzyme. The tryptophanyl-enzyme is destroyed during incubation with AMP or with pyrophosphate. The role of the tryptophanyl-enzyme as a possible intermediate in the course of aminoacylation of tRNATrp is discussed.

[Identification of the N-terminal peptide of bovine tryptophanyl-tRNA-synthetase]. / Identifikatsiia N-kontsevogo peptida bych'ei triptofanil-tRNK-sintetazy.

By means of covalent chromatography on thiopropyl-sepharose 6B the N-terminal, as well as other tryptic cysteine-containing peptides of the bovine tryptophanyl-tRNA-synthetase (EC 6.1.1.2) were purified and characterized, their structures being determined by a combination of plasma desorption mass spectrometry and peptide sequencing. In total, six different peptides containing seven cysteine residues were analysed. The N-terminal amino acid (presumably, alanine) was shown to be acetylated in the nature enzyme amino acid sequences of some cysteine-containing peptides proved to differ from those deduced from the cDNA structure, thus indicating the presence of the enzyme's isoforms. The purification does not affect the peptides' sulfhydryl groups. The number of cysteine residues in the peptides could be determined with a high accuracy by measuring their masses before and after alkylation with 4-vinylpyridine.

[Amino acid sequence of various peptides of tryptophanyl-tRNA-synthetase from bovine pancreas]. / Aminokislotnaia posledovatel'nost' nekotorykh peptidov triptofanil- tRNK-sintetazy iz podzheludochnoi zhelezy krupnogo rogatogo skota.

Method of isolation of the bovine pancreas tryptophanyl-tRNA synthetase is improved and a protein with greater than or equal to 99% purity, according to PAGE-SDS, is obtained. The pure enzyme is digested with clostripain and the hydrolysate is separated by FPLC anion-exchange chromatography followed by reversed phase HPLC. Amino acid sequences of 6 individual peptides, including C-terminal one, were determined by the automated Edman degradation. A peptide is also revealed which is encoded with the low degeneracy.

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.

Assignment of a gene for tryptophanyl-transfer ribonucleic acid synthetase (E.C. 6.1.1.2) to human chromosome 14.

We describe a simple method for locating tryptophanyl-tRNA synthetase (E.C. 6.1.1.2) on cellulose acetate gels (Cellogel) following electrophoresis. Employing electrophoretic conditions which result in the separation of mouse and human tryptophanyl-tRNA synthetase, we have analyzed extracts of a number of independently derived mouse-human somatic cell hybrids and subclones derived from these hybrids for the presence of human tryptophanyl-tRNA synthetase. Electrophoretic patterns of hybrid extracts which contain human tryptophanyl-tRNA synthetase exhibit three bands. This is consistent with published evidence that the enzyme for mammalian cells is a homologous dimer. The electrophoretic patterns derived from some hybrids are unusual in that the human and hybrid bands of activity are more intense than the mouse band from the same hybrid. An analysis of hybrid cells and extracts indicates that human tryptophanyl-tRNA synthetase segregates with human chromosome 14 and with the only enzyme marker which has previously been assigned to this chromosome, nucleoside phosphorylase.

Molecular and cellular studies of tryptophanyl-tRNA synthetase using monoclonal antibodies. Evaluation of a common antigenic determinant in eukaryotic, prokaryotic and archaebacterial enzymes which maps outside the catalytic domain.

Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl-tRNA synthetase were prepared. Am2 antibodies inhibit the Trp-tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40-kDa or 51-kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp-tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp-tRNA synthetase molecule. Am1 cross-reacts with Trp-tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis. A rapid two-step technique was developed for isolating electrophoretically homogeneous Trp-tRNA synthetase from Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaryotic Trp-tRNA synthetase, Am1 did not influence the activity of Trp-tRNA synthetase from E. coli. From the aforementioned results it follows that: (a) the conservation of part of the Trp-tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp-tRNA synthetases suggests that the dispensable part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNA(Trp) charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.

The plant aminoacyl-tRNA synthetases. Purification and characterization of valyl-tRNA, tryptophanyl-tRNA and seryl-tRNA synthetases from yellow-lupin seeds.

Valyl-tRNA, tryptophanyl-tRNA, and seryl-tRNA synthetases from yellow lupin seeds Lupinus luteus were purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on aminohexyl-Sepharose column and affinity chromatography on tRNA-Sepharose column. Valyl-tRNA synthetase consists of one polypeptide chain of molecular weight 125000 as judged by Sephadex G-200 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent. Seryl-tRNA synthetase, Mr equals 110000, is composed of two 55000-Mr subunits. Tryptophanyl-tRNA synthetase exhibits molecular weight of 200000 on Sephadex G-200 and 37000 in dodecylsulfate-polyacrylamide gel electrophoresis. This indicates that tryptophanyl-tRNA synthetase consists of several subunits (probably four). Since the seryl-tRNA synthetase exhibits the same mobility on dodecylsulfate-polyacrylamide gels both in the presence and absence of reducing agent it is concluded that there is no covalent bond(s) between the subunits of the enzyme. There is also no covalent bond(s) between the subunits of tryptophanyl-tRNA synthetase. Effect of anti-sulfhydryl reagents, monovalent salts, pH and different buffers on activity of the three synthetases is described. Kinetic constants for the substrates of the synthetases are also given. dATP is a substrate for seryl-tRNA synthetase but not for valyl-tRNA and tryptophanyl-tRNA synthetases.

Crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase containing enzymatically formed acyl transfer product tryptophanyl-ATP, an active site maker for the 3' CCA terminus of tryptophanyl-tRNATrp.

It has previously been shown that tryptophanyl-tRNA synthetase from Bacillus stearothermophilus crystallizes in different forms, depending on the substrates present during crystallization [Carter, C. W., Jr., & Carter, C. W. (1979) J. Biol. Chem. 254, 12219-12223]. Radiolabeling experiments show that the tetragonal crystals (type IV), grown in the presence of tryptophan and ATP, contain enzymatically formed 3'(2')-tryptophanyladenosine 5'-triphosphate (Trp-ATP). Trp-ATP is formed by acyl transfer of the tryptophanyl moiety of an acyladenylate intermediate, Trp-5'-AMP, to a second molecule of ATP bound in the site normally occupied by the 3' CCA terminus of tRNATrp. This compound is therefore a chemical marker in type IV crystals for that part of the tRNA binding site on the synthetase. Solution of this crystal structure, now in progress, may therefore provide useful information concerning the mechanism of aminoacylation of tRNATrp by this enzyme and may help locate its tRNA binding site.

Human tryptophan transfer ribonucleic acid synthetase. Composition, function of thiol groups, and structure of thiol peptides.

Human tryptophanyl-tRNA synthetase resembles its counterpart in Escherichia coli in quaternary structure (alpha2), but differs in molecular weight, amino acid composition, the number of thiol groups, and the relationship of the thiol groups to enzyme activity. Nevertheless, one of the thiol groups resides in a heptapeptide sequence homologous to a heptapeptide sequence containing a thiol group in the E. coli enzyme. Each subunit of the enzyme has 6 half-cystine residues, and four thiol groups are readily titrated with 5,5'-dithiobis(2-nitrobenzoic acid). Titration of these four thiol groups inactivates the enzyme, and the inactivation is partially reversible by reduction with dithiothreitol. One thiol group reacts rapidly unless L-tryptophan, ATP, and Mg2+ are present together.

High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.

The "eRF" clone corresponds to tryptophanyl-tRNA synthetase, not mammalian release factor.

To study the similarity between a putative cloned mammalian release factor (RF) and tryptophanyl-tRNA synthetase (TRS), a recombinant rabbit RF fusion protein was expressed from prokaryotic expression vectors. Purified fractions of the fusion proteins were tested for TRS and RF activities. Addition of the fusion protein to a TRS assay increased the binding of tryptophan to tRNA(Trp). However, in an assay for RF activity, the addition of the fusion protein resulted in release of only 1-3% of formylmethionine from an fMet-tRNA-AUG-ribosome intermediate that had been provided with UAAA as message. To confirm this result, the coding region of the putative eukaryotic RF clone "eRF" was used for in vitro transcription and translation in a rabbit reticulocyte lysate system, resulting in the synthesis of a single 56-kDa protein. The influence of this 56-kDa protein on the termination of translation directed by tobacco mosaic virus was studied. Tobacco mosaic virus RNA produced a major 126-kDa protein and a minor 184-kDa readthrough protein in an in vitro translation system. The protein generated from the "eRF" coding region did not inhibit biosynthesis of the 184-kDa readthrough virus protein. Instead, it increased the yield of both viral proteins. This increase was presumably due to its TRS activity. Chromatography of proteins derived from human lymphoblasts separated RF from TRS activity. Thus, our results indicate that the previously cloned "eRF" clone encodes TRS and that rabbit reticulocyte RF activity lies in a different protein.

Three G.C base pairs required for the efficient aminoacylation of tRNATrp by tryptophanyl-tRNA synthetase from Bacillus subtilis.

Acceptor stem is an essential region in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase. In this study, a library containing 20 nt random region and tryptophanyl-tRNA synthetase (TrpRS) from Bacillus subtilis were used for in vitro selection to find a new structural feature in the tRNA(Trp) acceptor stem sequence that is required for B. subtilis TrpRS recognition. After three rounds of selection, the TrpRS binding RNAs dominate the RNA pool. The aptamers share a common structure of three G.C base pairs, which was also found in the acceptor stem of wild-type B. subtilis tRNA(Trp). A series of tRNA(Trp) variants was prepared by in vitro transcription, and their efficiencies of tryptophanylation (k(cat)/K(M)) were measured with the aid of TrpRS from B. subtilis. The mutants that possess the three G.C base pairs and G73 discriminator base exhibit almost the same aminoacylation efficiencies as B. subtilis tRNA(Trp), while the G73 discriminator base itself cannot confer efficient aminoacylation to the tRNA(Trp) molecule. Thus, these three base pairs (G2.C71, G3.C70, and G4.C69) in the B. subtilis tRNA(Trp) acceptor stem were established to be new identity elements, and their importance was between the previously characterized major element G73 and minor elements A1/U72 and G5/C68. The minimum set of identity elements that is required to confer efficient aminoacylation by B. subtilis TrpRS included G73, G2.C71, G3.C70, and G4.C69.

Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase.

A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.