Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA

Transfer RNA can play a role in amino acid activation by aminoacyl-tRNA synthetases. For the prolyl-tRNA synthetase (ProRS) of Methanococcus jannaschii, which activates both proline and cysteine, the role of tRNA in amino acid selection and activation is of interest in the effort to understand the mechanism of the dual-specificity. While activation of proline does not require tRNA, whether or not tRNA is required in the activation of cysteine has been a matter of debate. Here, investigation of a series of buffer conditions shows that activation of cysteine occurs without tRNA in a wide-range of buffers. However, the extent of cysteine activation is strongly buffer-dependent, varying over a 180-fold range. In contrast, the extent of proline activation is much less sensitive to buffer conditions, varying over only a 36-fold range. We also find that addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual-specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

Zinc-mediated amino acid discrimination in cysteinyl-tRNA synthetase

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves a high level of amino acid specificity without an editing reaction. The crystal structure of CysRS bound to substrate cysteine suggested that direct thiol coordination to a tightly bound zinc ion at the base of the active site is the primary determinant of selectivity against non-cognate amino acids. This hypothesis has now been supported by spectroscopic studies of cobalt-substituted CysRS. Binding of cysteine, but not non-cognate amino acids, induces high absorption in the ligand-to-metal charge transfer region, providing evidence for formation of a metal-thiolate bond. In addition, mutations in the zinc ligands alter the absorption spectrum without reducing the discrimination against non-cognate amino acids. These results argue strongly for a major role for the zinc ion in amino acid discrimination by CysRS, where the tight zinc-thiolate interaction and the strict structural geometry of the metal ion are sufficient to reject serine by more than 20,000-fold at the binding step.     

Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl-tRNA synthetase lacking an editing domain

Aminoacyl-tRNA synthetases, a group of enzymes catalyzing aminoacyl-tRNA formation, may possess inherent editing activity to clear mistakes arising through the selection of non-cognate amino acid. It is generally assumed that both editing substrates, non-cognate aminoacyl-adenylate and misacylated tRNA, are hydrolyzed at the same editing domain, distant from the active site. Here, we present the first example of an aminoacyl-tRNA synthetase (seryl-tRNA synthetase) that naturally lacks an editing domain, but possesses a hydrolytic activity toward non-cognate aminoacyl-adenylates. Our data reveal that tRNA-independent pre-transfer editing may proceed within the enzyme active site without shuttling the non-cognate aminoacyl-adenylate intermediate to the remote editing site.     

Evolution of vitamin B2 biosynthesis. A novel class of riboflavin synthase in Archaea

The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment. Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer.     

Crystal structure of a non-discriminating glutamyl-tRNA synthetase

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.     

An Hfq-like protein in archaea: crystal structure and functional characterization of the Sm protein from Methanococcus jannaschii

The Sm and Sm-like proteins are conserved in all three domains of life and have emerged as important players in many different RNA-processing reactions. Their proposed role is to mediate RNA-RNA and/or RNA-protein interactions. In marked contrast to eukaryotes, bacteria appear to contain only one distinct Sm-like protein belonging to the Hfq family of proteins. Similarly, there are generally only one or two subtypes of Sm-related proteins in archaea, but at least one archaeon, Methanococcus jannaschii, encodes a protein that is related to Hfq. This archaeon does not contain any gene encoding a conventional archaeal Sm-type protein, suggesting that Hfq proteins and archaeal Sm-homologs can complement each other functionally. Here, we report the functional characterization of M. jannaschii Hfq and its crystal structure at 2.5 A resolution. The protein forms a hexameric ring. The monomer fold, as well as the overall structure of the complex is similar to that found for the bacterial Hfq proteins. However, clear differences are seen in the charge distribution on the distal face of the ring, which is unusually negative in M. jannaschii Hfq. Moreover, owing to a very short N-terminal alpha-helix, the overall diameter of the archaeal Hfq hexamer is significantly smaller than its bacterial counterparts. Functional analysis reveals that Escherichia coli and M. jannaschii Hfqs display very similar biochemical and biological properties. It thus appears that the archaeal and bacterial Hfq proteins are largely functionally interchangeable.     

Two tyrosine residues outside the editing active site in Giardia lamblia leucyl-tRNA synthetase are essential for the post-transfer editing

Leucyl-tRNA synthetase (LeuRS) is responsible for the Leu-tRNA^Leu synthesis. The connective peptide 1 (CP1) domain inserted into the Rossmann nucleotide binding fold possesses editing active site to hydrolyze the mischarged tRNA^Leu with noncognate amino acid, then to ensure high fidelity of protein synthesis. A few co-crystal structures of LeuRS with tRNA^Leu in different conformations revealed that tRNA^Leu 3′ end shuttled between synthetic and editing active sites dynamically with direct and specific interaction with the CP1 domain. Here, we reported that Y515 and Y520 outside the editing active site of CP1 domain of Giardia lamblia LeuRS (GlLeuRS) are crucial for post-transfer editing by influencing the binding affinity with mischarged tRNA^Leu. Mutations on Y515 and Y520 also decreased tRNA^Leu charging activity to various extents but had no effect on leucine activation. Our results gave some biochemical knowledge about interaction of tRNA^Leu 3′ end with the CP1 domain in archaeal/eukaryotic LeuRS.     

Amino acids activated by fengycin synthetase FenE

Fengycin is a lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis F29-3. Synthesis of this antibiotic requires five fengycin synthetases encoded by fenC, fenD, fenE, fenA, and fenB. In this study, we analyze the functions of the enzyme encoded by fenE, which contains two amino acid activation modules, FenE1 and FenE2. ATP-PP(i) exchange assay revealed that FenE1 activates l-Glu and FenE2 activates l-Ala, l-Val, and l-2-aminobutyric acid, indicating that FenE activates the fifth and the sixth amino acids in fengycin. Furthermore, l-Val is a better substrate than l-Ala for FenE2 in vitro, explaining why B. subtilis F29-3 normally produces twice as much of fengycin B than fengycin A, which contains d-Val and d-Ala at the sixth amino acid position, respectively. Results presented herein suggest that fengycin synthetase genes and amino acids in fengycin are colinear.     

Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii

In microorganisms and plants, mevalonate kinase is involved in the biosynthesis of isoprenoid derivatives, one of the largest groups of natural products. We subcloned the gene of mevalonate kinase from Methanococcus jannaschii into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. A variety of mutant expression plasmids including pMMK(R196K), pMMK(R196Q), pMMK(R196V), pMMK(K272R), and pMMK(K272A) have been constructed using site-directed mutagenesis. The wild-type protein and mutants were overexpressed and purified with a nickel HiTrap chelating metal affinity column to homogeneity. CD spectroscopy of wild-type protein and mutants indicates that none of the above mutations induces significant secondary structural changes. The results from kinetic studies showed that Arg196 is an essential residue for the function of the enzyme. Kinetic studies of Lys272 mutants indicate that salt bridge Lys272-Glu14 plays an important role in maintaining the active site microenvironment that is essential for catalytic activity of the enzyme.     

Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.     

Searching tRNA sequences for relatedness to aminoacyl-tRNA synthetase families

tRNA sequences were analyzed for sequence features correlated with known classes of aminoacyl-tRNA synthetase enzymes. The tRNAs were searched for distinguishing nucleotides anywhere in their sequences. The analyses did not find nucleotides predictive of synthetase class membership. We conclude that such nucleotides never existed in tRNA sequences or that they existed and were lost from many of the tRNA sequences during evolution.Based on a presentation made at a workshop—Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: H.B. Nicholas, Jr.     

The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA synthetase like activity

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5A using molecular replacement. The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS). It is much shorter than GluRS, which contains 468 residues, and lacks the complete domain interacting with the tRNA anticodon loop. As E.coli GluRS, YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain. The YadB cluster uses cysteine residues as the first three zinc ligands, but has a weaker tyrosine ligand at the fourth position. It shares with canonical amino acid RNA synthetases a major functional feature, namely activation of the amino acid (here glutamate). It differs, however, from GluRSs by the fact that the activation step is tRNA-independent and that it does not catalyze attachment of the activated glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA. These results suggest thus a novel function, distinct from that of GluRSs, for the yadB gene family.     

Molecular and functional dissection of a putative RNA-binding region in yeast mitochondrial leucyl-tRNA synthetase

Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the beta-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS-RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex.     

Methenyltetrahydrofolate synthetase is a high-affinity catecholamine-binding protein

Recombinant mouse 5,10-methenyltetrahydrofolate synthetase (MTHFS) was expressed in Escherichia coli and shown to co-purify with a chromophore that had a lambda(max) at 320nm. The chromophore remained bound to MTHFS during extensive dialysis, but dissociated from MTHFS when its substrate, 5-formyltetrahydrofolate, was bound. The chromophore was identified as an oxidized catecholamine by mass spectrometry and absorption spectroscopy. Purified recombinant mouse MTHFS and rabbit liver MTHFS proteins were shown to bind oxidized N-acetyldopamine (NADA) tightly. The addition of NADA to cell culture medium accelerated markedly folate turnover and decreased both folate accumulation and total cellular folate concentrations in MCF-7 cells. Expression of the MTHFS cDNA in MCF-7 cells increased the concentration of NADA required to deplete cellular folate. The results of this study are the first to identify a link between catecholamines and one-carbon metabolism and demonstrate that NADA accelerates folate turnover and impairs cellular folate accumulation in MCF-7 cells.     

Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence

While considerable progress has been achieved in plant CDPK studies in the past decade, there is relatively no information about the potential substrates of CRKs. In this report, a yeast two-hybrid screen was performed with truncated form of AtCRK3 as bait to identify its interacting proteins in an effort to dissect its physiological roles. One gene encoding cytosolic glutamine synthetase AtGLN1;1 was isolated. Further analyses indicated that AtGLN1;1 could interact specifically with AtCRK3 and the kinase domain of AtCRK3 and the catalytic domain of AtGLN1;1 were responsible for such interaction, respectively. Furthermore, in vitro and in vivo co-immunoprecipitation results strongly supported that they could physically interact with each other. Phosphorylation assays revealed that AtGLN1;1 could be specifically phosphorylated by AtCRK3 in vitro. All the results demonstrate that AtGLN1;1 may be a substrate of AtCRK3. In addition, both AtGLN1;1 and AtCRK3 could be induced by natural or artificially induced leaf senescence, implying that such interaction may be involved in the regulation of nitrogen remobilization during leaf senescence.     

A novel octameric AMP-forming acetyl-CoA synthetase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum

AMP-forming acetyl-CoA synthetases (ACSs) are ubiquitous in all three domains of life. Here, we report the first characterization of an ACS from a hyperthermophilic organism, from the archaeon Pyrobaculum aerophilum. The recombinant ACS, the gene product of ORF PAE2867, showed extremely high thermostability and thermoactivity at temperatures around 100 degrees C. In contrast to known monomeric or homodimeric mesophilic ACSs, the P. aerophilum ACS was a 610 kDa homooctameric protein, with a significant lower content of thermolabile (Cys, Asn, and Gln) and higher content of charged (Glu, Lys, and Arg) amino acids. Kinetic analyses revealed an unusual broad substrate spectrum for organic acids and an extremely high affinity for acetate (K(m) 3 microM).     

Effect of temperature and pressure on the proteolytic specificity of the recombinant 20S proteasome from<Emphasis Type="Italic"> Methanococcus jannaschii</Emphasis>

The hydrolytic specificity of the recombinant 20S proteasome from the deep-sea thermophile Methanococcus jannaschii was evaluated toward oxidized insulin B-chain across a range of temperatures (35°, 55°, 75°, and 90°C) and hydrostatic pressures (1, 250, 500, and 1,000 atm). Of the four temperatures considered, the same maximum overall hydrolysis rate was observed at both 55° and 75°C, which are much lower than the T_opt of 116°C previously observed for a small amide substrate (Michels and Clark 1997). At 35°C the rates of cleavage were highest at the carboxyl side of glutamine and leucine, whereas at the three higher temperatures, the most rapid cleavages occurred after leucine and glutamic acid residues. The distribution of proteolytic fragments and the cleavage sequence also varied between the lowest and higher temperatures. Application of hydrostatic pressure did not increase proteasome activity, as observed previously for the amide substrate (Michels and Clark 1997), but instead significantly reduced the overall conversion of the polypeptide substrate. Overall cleavage patterns observed for the recombinant M. jannaschii proteasome were similar to those reported previously for Thermoplasma acidophilum (Akopian et al. 1997) and human proteasomes (Dick et al. 1991), indicating that proteasome specificity has been conserved despite significant environmental diversity.Communicated by G. Antranikian