Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.

Control of 5',5'-dinucleoside triphosphate catabolism by APH1, a Saccharomyces cerevisiae analog of human FHIT.

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587-597, 1996) encodes a protein behaving in vitro as a dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5',5'-polyphosphates, with Ap3A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap3A-hydrolyzing activity and a 30-fold-elevated Ap3N concentration.

Comparison of the enzymatic properties of the two Escherichia coli lysyl-tRNA synthetase species.

In Escherichia coli, lysyl-tRNA synthetase activity is encoded by either a constitutive lysS gene or an inducible one, lysU. The two corresponding enzymes could be purified at homogeneity from a delta lysU and a delta lysS strain, respectively. Comparison of the pure enzymes, LysS and LysU, indicates that, in the presence of saturating substrates, LysS is about twice more active than LysU in the ATP-PPi exchange as well as in the tRNALys aminoacylation reaction. Moreover, the dissociation constant of the LysU-lysine complex is 8-fold smaller than that of the LysS-lysine complex. In agreement with this difference, the activity of LysU is less sensitive than that of LysS to the addition of cadaverine, a decarboxylation product of lysine and a competitive inhibitor of lysine binding to its synthetase. This observation points to a possible useful role of LysU, under physiological conditions causing cadaverine accumulation in the bacterium. Remarkably, these conditions also induce lysU expression. Homogeneous LysU and LysS were also compared in Ap4A synthesis. LysU is only 2-fold more active than LysS in the production of this dinucleotide. This makes unlikely that the heat-inducible LysU species could be preferentially involved in the accumulation of Ap4A inside stressed Escherichia coli cells. This conclusion could be strengthened by determining the concentrations of Ap4N (N = A, C, G, or U) in a delta lysU as well as in a lysU+ strain, before and after a 1-h temperature shift at 48 degrees C. The measured concentration values were the same in both strains.

Properties of the lysyl-tRNA synthetase gene and product from the extreme thermophile Thermus thermophilus.

A DNA region carrying lysS, the gene encoding the lysyl-tRNA synthetase, was cloned from the extreme thermophile prokaryote Thermus thermophilus VK-1 and sequenced. The analysis indicated an open reading frame encoding a protein of 492 amino acids. This putative protein has significant homologies to previously sequenced lysyl-tRNA synthetases and displays the three motifs characteristic of class II aminoacyl-tRNA synthetases. The T. thermophilus lysS gene was overexpressed in Escherichia coli by placing it downstream of the E. coli beta-galactosidase gene promoter on plasmid pBluescript and by changing the ribosome-binding site. The overproduced protein was purified by heat treatment of the crude extract followed by a single anion-exchange chromatography step. The protein obtained is remarkably thermostable, retaining nearly 60% of its initial tRNA aminoacylation activity after 5 h of incubation at 93 degrees C. Finally, lethal disruption of the lysRS genes of E. coli could not be compensated for by the addition in trans of the T. thermophilus lysS gene despite the fact that this gene was overexpressed and that its product specifically aminoacylates E. coli tRNA(Lys) in vitro.

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).

Pyrophosphatase is essential for growth of Escherichia coli.

The ppa gene for inorganic pyrophosphatase is essential for the growth of Escherichia coli. A recombinant with a chromosomal ppa::Kanr lesion and a temperature-sensitive replicon with a ppa+ gene showed a temperature-sensitive growth phenotype, and a mutant with the sole ppa+ gene under control of the lac promoter showed inducer-dependent growth. When the lacp-ppa mutant was subcultured without inducer, the pyrophosphatase level decreased, the PPi level increased, and growth stopped. Cellular PPi reached 16 mM about 6 h after growth arrest without loss of cell viability.

In vivo synthesis of adenylylated bis(5'-nucleosidyl) tetraphosphates (Ap4N) by Escherichia coli aminoacyl-tRNA synthetases.

The role of aminoacyl-tRNA synthetases in the in vivo synthesis of adenylylated bis(5'-nucleosidyl) tetraphosphates (Ap4N) was studied by measuring the concentration of these nucleotides in Escherichia coli cells overproducing lysyl-, methionyl- phenylalanyl-, or valyl-tRNA synthetase. Overproduction of each aminoacyl-tRNA synthetase (20- to 80-fold) was accompanied by a significant increase in intracellular Ap4N concentration (3- to 14-fold). As expected, non-adenylylated bis(5'-nucleosidyl) tetraphosphate concentration was not changed by synthetase overproduction. It was also verified that overproduction of an inactive methionyl-tRNA synthetase mutant did not modify Ap4N concentration. Ap4N accumulation during heat shock occurred in all strains studied. The increase factor (approximately 50-fold after 1 hr at 48 degrees C) was not changed by overproduction of any of the aminoacyl-tRNA synthetases studied, including that of the heat-inducible form of lysyl-tRNA synthetase from the lysU gene. Together, these results establish that aminoacyl-tRNA synthetases are involved in Ap4N biosynthesis during exponential growth as well as during heat shock.

Non-adenylylated bis(5'-nucleosidyl) tetraphosphates occur in Saccharomyces cerevisiae and in Escherichia coli and accumulate upon temperature shift or exposure to cadmium.

A new set of bis(5'-nucleosidyl) tetraphosphates, the Bp4B' nucleotides (B and B' = C, G, or U not equal to A), are demonstrated in living cells. In exponentially growing Saccharomyces cerevisiae, cellular concentrations of Cp4U, Up4U, Gp4G, Cp4C, Gp4U, and Gp4C are 210, 200, 60, 50, 40, and 30 nM, respectively. It is likely that these nucleotides originate from the action of diadenosine-5',5"'-P1,P4-tetraphosphate alpha,beta-phosphorylase, an enzyme recently found in yeast. Upon temperature shift or exposure to cadmium, the Bp4B' nucleotides strongly accumulate in the yeast cells. In Escherichia coli, the same nucleotides occur, and similar effects of temperature shift or of cadmium are observed. However, in the bacterium, the origin of these nucleotides is not known. To quantitate these nucleotides in cellular extracts, specific procedures were developed. In the first step, after purification of the mixture of Np4N' (N and N' = A, C, G, or U) nucleotides, the Ap4N nucleotides are specifically removed by incubation with lysyl-tRNA synthetase. In the second step, the Bp4B' species are resolved with the help of anion-exchange high performance liquid chromatography. In the third step, the concentration of each Bp4B' is measured using three coupled enzymatic reactions to produce ATP and bioluminescence. With this strategy, 0.01 pmol of any Bp4B' nucleotide can be reliably detected.

Yeast diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase.

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.

Variation of Ap4A and other dinucleoside polyphosphates in stressed Drosophila cells.

The unusual bis(5'-nucleosidyl)oligophosphates: Ap4A, Ap4G, Ap3A, and Ap3G, have been measured in cultures of Drosophila cells. Exponentially growing cells contain concentrations of 0.25, 0.31, 0.87, and 2.25 microM, respectively. These nucleotides have been followed after stressing the cells either by CdCl2 addition or by heat-shock treatment. Their concentrations are not affected by exposure to 500 microM CdCl2 during 6 h. Beyond this threshold of cadmium concentration, the nucleotides increase. With 5 mM CdCl2, an enhancement by 2 orders of magnitude of all the dinucleoside tri- and tetra-phosphates is observed. Upon heat-shock from 19 to 37 degrees C, Ap4A, Ap3A, and Ap3G increase up to 2.2, 3, and 3.3 times their initial levels, respectively. The increase is achieved within 1 h.

Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase.

Diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells. It produces 2.4 mg of homogeneous enzyme with a yield of 3.1%. The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 [37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2]. The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography. Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not). Among the products, a nucleoside diphosphate is always formed. ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is. Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 microM MnCl2, the stimulation is 900-fold. Ca2+, Fe2+, and Mg2+ have no effect. The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+. The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP. However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+. Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of zinc in 5',5'-diadenosine tetraphosphate production by aminoacyl-transfer RNA synthetases.

Aminoacyl-tRNA synthetases are capable of converting 5'-ATP into 5',5'-diadenosine tetraphosphate. The reaction reflects the reversal of enzyme-bound aminoacyl-adenylate by ATP instead of PPi. In the case of a few prokaryotic as well as eukaryotic aminoacyl-tRNA synthetases, the initial rate of diadenosine tetraphosphate synthesis can be greatly enhanced upon adding small amounts of zinc. This observation enables us to establish a relationship between diadenosine tetraphosphate, a nucleotide possibly involved in controlling cell proliferation, and a metallic cofactor, which is believed to play a role in tumour growth.

Zinc-dependent synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNA synthetases.

Zinc greatly stimulates the initial rate of in vitro synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNMA synthetases. In the case of each enzyme, maximum stimulation of 50- to 100-fold is reached upon addition of 50 microM ZnC2 to a reaction mixture (37 degrees c, pH 7.8) containing 50 microM dithioerythritol, 150 mM KCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM cognate L-aminoacid, catalytic amounts of the aminoacyl-tRNA synthetase, and unlimiting pyrophosphatase activity. This observation made with aminoacyl-tRNA synthetases of mammalian origin supports the proposal that changes in cellular free zinc ion concentration could contribute to 5',5'-diadenosine tetraphosphate variations in animal cells as a function of growth activity.

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. I. Species specificity of the polypeptide composition.

Using a three-step procedure designed to minimize the risks of proteolysis, high molecular weight complexes containing the same seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, lysine, arginine, glutamic acid, and glutamine were purified from sheep liver and spleen, as well as from rabbit reticulocytes and liver. The polypeptide composition of these complexes, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is characteristic of the animal species from which they are derived. The complexes from sheep liver and spleen display indistinguishable polypeptide patterns composed of 11 major components. Of the 10 common components which characterize the complexes of rabbit reticulocytes and liver, 4 are also shared by the complexes from sheep, while 6 have distinctly different electrophoretic mobilities. Furthermore, in the case of the complex from rabbit reticulocytes, it is shown that the enzyme and polypeptide composition of the complex is independent of the purification method employed. The isolation of high molecular weight complexes of identical aminoacyl-tRNA synthetase and polypeptide compositions from two cell types as radically different as rabbit reticulocytes and hepatocytes suggests that these multienzyme complexes do not arise as artifacts of preparation and supports the view that they reflect a structural organization existing within the cell.

Macromolecular complex of aminoacyl-tRNA synthetases from sheep liver. Identification of the methionyl-tRNA synthetase component by affinity labeling.

Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet. The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost. Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet. The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex.

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).

Comparative structural analysis of myosin after limited tryptic hydrolysis by use of two-dimensional gel electrophoresis.

Polypeptides obtained by limited tryptic digestion of several myosins have been analyzed by a two-dimensional electrophoresis technique. The different maps thus obtained exhibit some common and distinct features specific of the myosins studied. Myosins from rabbit, fast and slow muscle and cardiac tissue, as well as beef cardiac myosin, have been compared. The polypeptides obtained vary in molecular weight from 120 000 60 15 000. The light chains LC1 and LC2, have disappeared. A peptide which comigrates with fast type LC3 is however found. The fast myosin hydrolyzate is very different from that obtained by the hydrolysis of slow and cardiac myosin. Numerous peptides are common to cardiac and slow myosins. However a few peptides are specific of the two myosin types. In the hydrolyzate from fetal calf myosin, some of the typical slow, fast and cardiac peptides can be found. However several apparently unique fetal peptides are also present. The comparison of the fetal calf tissue myosin hydrolyzate and that of [35S] methionine labeled myosin from myotubes in cultures shows qualitatively a very great homology. Thus the protein synthesized by cultured cells seems to be very similar to or the same as that of the embryonic tissue.

Methionyl-tRNA synthetase from sheep liver. Purification of a fully active monomer derived from high-molecular-weight complexes by trypsin treatment. Evidence for immunological cross-reaction with the corresponding enzyme from sheep mammary gland.

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.

[Identification of an essential glutamate residue in the 3-phosphoglycerate kinase of yeast]. / Identification du groupement glutamique essentiel de la 3-phosphoglycérate kinase de levure.

The incorporation of nitrotyrosine into 3-phosphoglycerate kinase activated by carbodiimide results in the chemical modification of a single essential residue. After total proteolytic digestion, isolation of the dipeptide gamma Glu-NO2 Tyr by immuno-affinity chromatography indicates the implication of a glutamyl residue. It is interesting to point out the applicability of the method described for the purification of peptides containing carboxyl residues.