Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.     

The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.     

The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues

S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues.     

S-Adenosylmethionine synthetase from Escherichia coli

Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site.     

Aminoacylation of tRNAs as critical step of protein biosynthesis.

Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.     

Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl-tRNA synthetase

Sequence comparisons among methionyl-tRNA synthetases from different organisms reveal only one block of homology beyond the last beta strand of the mononucleotide fold. We have introduced a series of semi-conservative amino acid replacements in the conserved motif of yeast methionyl-tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side-chains with the tRNA anticodon.     

Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis.

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.     

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.     

Tripolyphosphatase associated with S-adenosylmethionine synthetase isozymes from rat liver

S-Adenosylmethionine (AdoMet) synthetase alpha and beta were purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis from rat liver. When the purified enzymes were applied onto Sephacryl S-200, each synthetase was eluted together with a tripolyphosphatase. The activities of these isozymes in synthesizing AdoMet and in hydrolyzing tripolyphosphate decreased in parallel with increasing amounts of rabbit anti-(beta-form) IgG. The activity of the beta-form isozyme was markedly stimulated by the addition of tripolyphosphate, whereas that of the alpha-form isozyme was inhibited. The tripolyphosphatase activity of both the alpha- and the beta-form was markedly stimulated by the addition of AdoMet. The tripolyphosphatases of each isozyme showed some other similar properties.     

Mapping of biologically relevant sites on human IL-1 beta using monoclonal antibodies

mAb have been raised that recognize human IL-1 beta. Using overlapping peptide fragments expressed in yeast and bacteria, we have mapped the regions of the protein to which these antibodies bind. To assess the relevance of the different regions of IL-1 beta for the expression of its biologic activity, the ability of the antibodies to block IL-1 activity was assayed. Antibodies recognizing the regions 133-148 and 251-269 of human IL-1 beta could inhibit the activity of IL-1 beta, but not of IL-1 alpha, in two different biologic assays, the murine thymocyte proliferation and PGE2 release from human fibroblasts. Conversely, antibodies that recognize the region 218-243 have only a moderate inhibitory effect on the IL-1 beta biologic activity in both assays. Finally, an antibody mapping to the region 148-192 did not inhibit IL-1 beta activity either on thymocytes or on fibroblasts. It is suggested that IL-1 beta-induced cell activation involves different regions of the protein and that both N-terminal and C-terminal fragments are involved in the correct functioning of the IL-1 beta molecule.     

Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E. coli AlkA protein. The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E. coli or yeast DNA. A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence. Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs. However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E. coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively. Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells. A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.     

Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases

Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat

The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA. The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme. The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39. The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast. The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E. coli and yeast enzymes. The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E. coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes. Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA. This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein. Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.     

Solvent-accessible cysteines in human cystathionine beta-synthase: crucial role of cysteine 431 in S-adenosyl-L-methionine binding

Cystathionine beta-synthase (CBS) is a tetrameric heme protein that catalyzes the PLP-dependent condensation of serine and homocysteine to cystathionine. CBS occupies a crucial regulatory position between the methionine cycle and transsulfuration. Human CBS contains 11 cysteine residues that are highly conserved in mammals but completely absent in the yeast enzyme, which catalyzes an identical reaction, suggesting a possible regulatory role for some of these residues. In this report, we demonstrate that in both the presence and absence of the CBS allosteric regulator S-adenosyl-l-methionine (AdoMet), only C15 and C431 of human CBS are solvent accessible. Mutagenesis of C15 to serine did not affect catalysis or AdoMet activation but significantly reduced aggregation of the purified enzyme in vitro. Mutagenesis of C431 resulted in a constitutively activated form of CBS that could not be further activated by either AdoMet or thermal activation. We and others have previously reported a number of C-terminal CBS point mutations that result in a decreased or abolished response to AdoMet. In contrast to all of these previously investigated CBS mutants, the C431 mutant form of CBS was unable to bind AdoMet, indicating that either this residue is directly involved in AdoMet binding or its absence induces a conformational change that destroys the integrity of the binding site for this regulatory ligand.     

The primary structure of human glutaminyl-tRNA synthetase. A highly conserved core, amino acid repeat regions, and homologies with translation elongation factors

We describe the nucleotide sequences of several overlapping cDNA clones specific for human glutaminyl-tRNA synthetase. The identified open reading frame indicates that the enzyme is composed of 1440 amino acids. A stretch of about 360 amino acids of the human enzyme is highly conserved in bacterial and yeast glutaminyl-tRNA synthetases. However, the human enzyme is three times larger than the bacterial and twice as large as the yeast enzyme suggesting that a considerable part of human glutaminyl-tRNA synthetase has evolved to perform functions other than the charging of tRNA. The sequence outside of the conserved core region includes three 57-amino acid repeats followed by a consecutive stretch of 11 charged amino acids. A computer assisted search of two protein data banks reveals that the human glutaminyl-tRNA synthetase shares small blocks of amino acid similarities with several other synthetases of different amino acid specificities. Interestingly, the enzyme also possesses some regions of similarities with eukaryotic translation elongation factor EF-1 but not with any other sequence stored in the protein data banks. The coding regions of human and mouse glutaminyl-tRNA synthetase cDNAs are identical at 94% of the codons. However, the 3'-noncoding regions of mouse and human mRNAs are more divergent (approximately 68%) but both possess the potential to form stable secondary structures of similar general architecture.     

Structure and evolution of a group of related aminoacyl-tRNA synthetases

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.     

Sterol methyltransferase: functional analysis of highly conserved residues by site-directed mutagenesis

Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity. Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity. Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity. Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding. Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9. These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction. Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments. Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation.