Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.     

Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii

Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell.     

Kinetic characterization of adenylosuccinate synthetase from the thermophilic archaea Methanocaldococcus jannaschii

Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.     

Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNA(Pro) acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar-phosphate backbone interactions in recognition of human tRNA(Pro). Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2'-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.     

Crystal structure and function of 5-formaminoimidazole-4-carboxamide ribonucleotide synthetase from Methanocaldococcus jannaschii

Purine biosynthesis requires 10 enzymatic steps in higher organisms, while prokaryotes require an additional enzyme for step 6. In most organisms steps 9 and 10 are catalyzed by the purH gene product, a bifunctional enzyme with both 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) synthase and inosine monophosphate (IMP) cyclohydrolase activity. Recently it was discovered that Archaea utilize different enzymes to catalyze steps 9 and 10. An ATP-dependent FAICAR synthetase is encoded by the purP gene, and IMP cyclohydrolase is encoded by the purO gene. We have determined the X-ray crystal structures of FAICAR synthetase from Methanocaldococcus jannaschii complexed with various ligands, including the tertiary substrate complex and product complex. The enzyme belongs to the ATP grasp superfamily and is predicted to use a formyl phosphate intermediate formed by an ATP-dependent phosphorylation. In addition, we have determined the structures of a PurP orthologue from Pyrococcus furiosus, which is functionally unclassified, in three crystal forms. With approximately 50% sequence identity, P. furiosus PurP is structurally homologous to M. jannaschii PurP. A phylogenetic analysis was performed to explore the possible role of this functionally unclassified PurP.     

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a k_cat/K_M of 3.2 × 10⁷ and 3.0 × 10⁷ s^{? 1}M^{? 1}, respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site‐directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10‐fold decrease in k_cat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828–840. © 2016 Wiley Periodicals, Inc.     

Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown

Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. S?ll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNA(Cys) formation in M. jannaschii still remains to be discovered.     

Palindromic repetitive DNA elements with coding potential in Methanocaldococcus jannaschii

We have identified 141 novel palindromic repetitive elements in the genome of euryarchaeon Methanocaldococcus jannaschii. The total length of these elements is 14.3kb, which corresponds to 0.9% of the total genomic sequence and 6.3% of all extragenic regions. The elements can be divided into three groups (MJRE1-3) based on the sequence similarity. The low sequence identity within each of the groups suggests rather old origin of these elements in M. jannaschii. Three MJRE2 elements were located within the protein coding regions without disrupting the coding potential of the host genes, indicating that insertion of repeats might be a widespread mechanism to enhance sequence diversity in coding regions.     

Structure and activity of an aminoacyl-tRNA synthetase that charges tRNA with nitro-tryptophan

The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNA(Trp) with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.     

Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus(Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.     

Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex

Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.     

Mammalian prolyl-tRNA synthetase corresponds to the approximately 150 kDa subunit of the high-M(r) aminoacyl-tRNA synthetase complex.

The high-M(r) aminoacyl-tRNA synthetase complex previously purified from sheep liver differed from those isolated from several other mammalian sources by the absence of prolyl-tRNA synthetase activity and the presence of glutamyl tRNA synthetase as a polypeptide of 85 kDa instead of 150 kDa. Using a milder extraction procedure that minimizes proteolysis, we now report the isolation of a sheep liver complex that contains both prolyl-tRNA synthetase activity and the 150-kDa polypeptide. The correspondence between prolyl-tRNA synthetase and the 150-kDa polypeptide, inferred from the results of several approaches reported in this study, was further demonstrated by showing that antibodies to a free form of sheep liver prolyl-tRNA synthetase generated by endogenous proteolysis, specifically reacted with the 150-kDa components of the complexes from sheep and rabbit, but failed to react with the previously purified complex from sheep that contained neither prolyl-tRNA synthetases activity nor the 150-kDa component. Moreover, we show that the 150-kDa polypeptide is also recognized by antibodies to the 85-kDa polypeptide previously assigned to glutamyl-tRNA synthetase. The possibility that the largest subunit of the mammalian high-M(r) complexes may be a bifunctional protein encoding both glutamyl- and prolyl-tRNA synthetase activities is considered and discussed in light of the recently published sequence of the corresponding polypeptide from HeLa cells. In accordance with this prediction, we show that the amino acid sequence of the carboxyl-terminal moiety of this bifunctional polypeptide shows significant similarity to the sequence of prolyl-tRNA synthetase from Escherichia coli.