Effects of amino acids on Thiobacillus acidophilus. II. Threonine deaminase

Biosynthetic L-threonine deaminase was partially purified 73-fold with a 60% recovery from Thiobacillus acidophilus by ammonium sulfate fractionation and by Sepharose 6B-C1 chromatography. The optimal pH for enzyme activity was between 9.0 and 10.0 and no optimal pH shift was observed in the presence of L-isoleucine, an inhibitor. The enzyme was effectively inhibited by L-isoleucine and showed homotropic interaction only in the presence of L-isoleucine. Kinetic studies indicate that there are at least two threonine binding sites and at least two isoleucine binding sites. The Km for threonine is 2.5 x 10(-3) M. The inhibition due to isoleucine is reversed by low concentrations of L-valine. L-Valine at high concentration acts as a substrate analogue and competitively inhibits L-threonine binding at the active site; the K1 is 1.6 x 10(-2) M.     

Alterations in the excess synthesis of riboflavin in Pichia guilliermondii under the influence of branched-chain amino acids

The effect of the branched-chain amino acids: L-valine, L-isoleucine and L-leucine on riboflavin overproduction was studied in the Pichia (Candida) guilliermondii (Cast.) Lang. et G. yeast, L-Val, L-Ile and L-Leu were found to inhibit riboflavin overproduction only under iron-deficient growth conditions. Other amino acids used did not show this effect. In crude extracts of P. guilliermondii the specific activity of the alpha-acetolactate forming enzyme, pH 8.0, is inhibited by L-Val. It is revealed that the activity of alpha-acetolactate synthetase in iron-deficient riboflavin-overproduction cells was exceedingly higher than in the valine-inhibited cells. Under iron deficiency alpha-acetolactate synthetase shows maximal activity after 48 h of growth. It was possible to detect diacetyl (and aceton) in the culture fluid.     

Switching the amino acid specificity of an aminoacyl-tRNA synthetase

The accuracy of protein synthesis essentially rests on aminoacyl-tRNA synthetases that ensure the correct attachment of an amino acid to the cognate tRNA molecule. The selection of the amino acid substrate involves a recognition stage generally followed by a proofreading reaction. Therefore, to change the amino acid specificity of a synthetase in the aminoacylation reaction, it is necessary to alleviate the molecular barriers which contribute its editing function. In an attempt to accommodate a noncognate amino acid into the active site of a synthetase, we chose a pair of closely related enzymes. The current hypothesis designates glutaminyl-tRNA synthetase (GlnRS) as a late component of the protein synthesis machinery, emerging in the eukaryotic lineage by duplication of the gene for glutamyl-tRNA synthetase (GluRS). By introducing GluRS-specific features into the Rossmann dinucleotide-binding domain of human GlnRS, we constructed a mutant GlnRS which preferentially aminoacylates tRNA with glutamate instead of glutamine. Our data suggest that not only the transition state for aminoacyl-AMP formation but also the proofreading site of GlnRS are affected by that mutation.     

Mutational analysis of a leucine heptad repeat motif in a class I aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases activate amino acids with ATP to form aminoacyl adenylates as the essential intermediates for aminoacylation of their cognate tRNAs. The class I Escherichia coli cysteine tRNA synthetase contains an N-terminal nucleotide binding fold that provides the catalytic site of adenylate synthesis. The C-terminal domain of the cysteine enzyme is predominantly alpha-helical and contains a leucine heptad repeat motif. We show here that specific substitutions of leucines in the leucine heptad repeats reduced tRNA aminoacylation. In particular, substitution of Leu316 with phenylalanine reduced the catalytic efficiency of aminoacylation by 1000-fold. This deleterious effect was partially alleviated by a more conservative substitution of leucine with valine. Filter binding assays show that neither the phenylalanine nor the valine substitution at Leu316 had a major effect on the ability of the cysteine enzyme to bind tRNA(Cys). In contrast, pyrophosphate exchange assays show that both substitutions decreased the adenylate synthesis activity of the enzyme. Analysis of these results suggests that the primary defect of the valine substitution is executed at adenylate synthesis while that of the phenylalanine substitution is at both adenylate synthesis and the transition state of tRNA aminoacylation. Thus, although Leu316 is located in the C-terminal domain of the cysteine enzyme, it may modulate the capacity of the N-terminal domain for amino acid activation and tRNA aminoacylation through a domain-domain interaction.     

L-arginine recognition by yeast arginyl-tRNA synthetase

The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.     

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-A resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Omega loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Mutational analysis suggests the same design for editing activities of two tRNA synthetases

Although the structural basis for amino acid activation by class I tRNA synthetases is known, that for their editing activities has remained elusive. Two class I tRNA synthetases discriminate closely similar amino acids by RNA-independent and RNA-dependent mechanisms. In the absence of tRNA, isoleucyl-tRNA synthetase misactivates valine, while valyl-tRNA synthetase misactivates threonine. Both enzymes improve amino acid discrimination by tRNA-dependent hydrolytic editing reactions. Recent mutational analysis of an isoleucyl-tRNA synthetase showed that discrimination of valine from isoleucine by amino acid activation was functionally independent of discrimination by editing. In this work, we used mutational analysis to test whether the two types of amino acid discrimination were functionally independent in valyl-tRNA synthetase. We obtained four mutations in the valine enzyme which severely affected amino acid activation. The two most defective enzymes reduced kcat/Km for activation of valine by more than 4 orders of magnitude and were essentially inactive for aminoacylation. These two defective enzymes were tested and found to be unaltered in catalysis of rapid and selective removal of threonine misacylated onto valine tRNA. On the basis of these data, and in spite of there being few residues conserved between the two proteins in a region believed important for editing, we propose that the valine and isoleucine enzymes share a global design which functionally separates amino acid editing from amino acid activation.     

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.     

Species-specific differences in the operational RNA code for aminoacylation of tRNAPro

An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.     

Current approaches to the use of radiotherapy in patients with non-small-cell lung cancer

Interactions of specific amino acid residues of the carboxyl-terminal domain of MetRS with the CAU anticodon of tRNAMet assure accurate and efficient aminoacylation. The substitution of one such residue, Trp461 by Phe, impairs the binding of cognate tRNA, but enhances the binding of noncognate tRNAs, particularly those containing G at the wobble position. However, the enhanced binding of noncognate tRNAs is not accompanied by the increased aminoacylation of these tRNAs. A genetic screening procedure was designed to isolate methionyl-tRNA synthetase mutants which were able to aminoacylate a GGU (threonine) anticodon derivative of tRNAfMet. One such mutant, obtained from W461F MetRS, had an Ile29 to Thr substitution in helix A located in the amino-terminal dinucleotide-fold domain that forms the site for amino acid activation. Analysis of the catalytic properties of the I29T/W461F enzyme indicates that the mutation in helix A of the dinucleotide-fold domain affects kcat for aminoacylation of tRNAs having a GGU threonine anticodon. Interactions with cognate tRNAfMet (CAU), as well as with methionine and ATP were not affected by the Ile29 to Thr substitution. We conclude that the I29T substitution leads to a slight adjustment of the alignment of the CCA stem of noncognate tRNAs (GGU) in the catalytic domain of the enzyme, reflected in the increase in kcat, which also allows mischarging in vivo. A function of Ile29 is therefore to minimize the mischarging of tRNAThr (GGU) by methionyl-tRNA synthetase. The methods described here provide useful tools for examining the mechanisms of tRNA selection by aminoacyl-tRNA synthetases.     

Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis

The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.     

Effect of Rare Earth Ions on Kinetic Properties of Escherichia coli Leucyl-tRNA Synthetase

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Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit

We show here that the class I human cytoplasmic isoleucyl-tRNA synthetase is an exceptionally large polypeptide (1266 aa) which, unlike its homologues in lower eukaryotes and prokaryotes, has a third domain of two repeats of an approximately 90-aa sequence appended to its C-terminal end. While extracts of Escherichia coli do not aminoacrylate mammalian tRNA with isoleucine, expression of the cloned human gene in E. coli results in charging of the mammalian tRNA substrate. The appended third domain is dispensable for detection of this aminoacylation activity and may be needed for assembly of a multisynthetase complex in mammalian cells. Alignment of the sequences of the remaining two domains shared by isoleucyl-tRNA synthetases from E. coli to human reveals a much greater selective pressure on the domain needed for tRNA acceptor helix interactions and catalysis than on the domain needed for interactions with the anticodon. This result may have implications for the historical development of an operational RNA code for amino acids.     

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.     

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.     

Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C

Phosphoinositide-specific phospholipases C (PI-PLCs) are ubiquitous enzymes that catalyse the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol (DAG). Whereas the eukaryotic PI-PLCs play a central role in most signal transduction cascades by producing two second messengers, inositol-1,4,5-trisphosphate and DAG, prokaryotic PI-PLCs are of interest because they act as virulence factors in some pathogenic bacteria. Bacterial PI-PLCs consist of a single domain of 30 to 35 kDa, while the much larger eukaryotic enzymes (85 to 150 kDa) are organized in several distinct domains. The catalytic domain of eukaryotic PI-PLCs is assembled from two highly conserved polypeptide stretches, called regions X and Y, that are separated by a divergent linker sequence. There is only marginal sequence similarity between the catalytic domain of eukaryotic and prokaryotic PI-PLCs. Recently the crystal structures of a bacterial and a eukaryotic PI-PLC have been determined, both in complexes with substrate analogues thus enabling a comparison of these enzymes in structural and mechanistic terms. Eukaryotic and prokaryotic PI-PLCs contain a distorted (beta alpha)8-barrel as a structural motif with a surprisingly large structural similarity for the first half of the (beta alpha)8-barrel and a much weaker similarity for the second half. The higher degree of structure conservation in the first half of the barrel correlates with the presence of all catalytic residues, in particular two catalytic histidine residues, in this portion of the enzyme. The second half contributes mainly to the features of the substrate binding pocket that result in the distinct substrate preferences exhibited by the prokaryotic and eukaryotic enzymes. A striking difference between the enzymes is the utilization of a catalytic calcium ion that electrostatically stabilizes the transition state in eukaryotic enzymes, whereas this role is filled by an analogously positioned arginine in bacterial PI-PLCs. The catalytic domains of all PI-PLCs may share not only a common fold but also a similar catalytic mechanism utilizing general base/acid catalysis. The conservation of the topology and parts of the active site suggests a divergent evolution from a common ancestral protein.     

A stringent test for the nucleotide switch mechanism of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. The X-ray crystal structure of the enzyme has revealed that the two nucleotide binding sites are separated by approximately 35 A. Isotopic oxygen exchange of 18O and 16O between solvent water and [13C]bicarbonate was measured using 13C NMR spectroscopy during substrate turnover in the presence and absence of glutamine as a nitrogen source. In the absence of added glutamine, CPS catalyzed the exchange of one oxygen atom from bicarbonate with solvent water during every turnover of the bicarbonate-dependent ATPase reaction. In the presence of added glutamine, there was no exchange of solvent water with bicarbonate during the enzymatic synthesis of carbamoyl phosphate, indicating that any carbon-containing intermediate in the reaction mechanism is committed to the formation of carbamoyl phosphate and is not subject to hydrolysis. These results are fully consistent with a chemical mechanism that requires the physical migration of the carbamate intermediate from the site of its formation within one of the nucleotide binding domains to the other nucleotide binding domain for subsequent phosphorylation by the second MgATP. These results are not compatible with a nucleotide switch mechanism. The nucleotide switch mechanism includes the synthesis of carbamoyl phosphate entirely within a single nucleotide binding domain and concurrent conformational changes driven by the bicarbonate-dependent hydrolysis of MgATP at the second nucleotide binding domain.     

Sequence divergence of seryl-tRNA synthetases in archaea

The genomic sequences of Methanococcus jannaschii and Methanobacterium thermoautotrophicum contain a structurally uncommon seryl-tRNA synthetase (SerRS) sequence and lack an open reading frame (ORF) for the canonical cysteinyl-tRNA synthetase (CysRS). Therefore, it is not clear if Cys-tRNACys is formed by direct aminoacylation or by a transformation of serine misacylated to tRNACys. To address this question, we prepared SerRS from two methanogenic archaea and measured the enzymatic properties of these proteins. SerRS was purified from M. thermoautotrophicum; its N-terminal peptide sequence matched the sequence deduced from the relevant ORF in the genomic data of M. thermoautotrophicum and M. jannaschii. In addition, SerRS was expressed from a cloned Methanococcus maripaludis serS gene. The two enzymes charged serine to their homologous tRNAs and also accepted Escherichia coli tRNA as substrate for aminoacylation. Gel shift experiments showed that M. thermoautotrophicum SerRS did not mischarge tRNACys with serine. This indicates that Cys-tRNACys is formed by direct acylation in these organisms.