The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is essential for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNAs. The aaRSs are essential for coupling the correct amino acid and tRNA molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond translation. Multiprotein complexes containing aaRSs are found in all three domains of life playing roles in splicing, apoptosis, viral assembly, and regulation of transcription and translation. An overview of the complexes aaRSs form in all domains of life is presented, demonstrating the extensive network of connections between the translational machinery and cellular components involved in a myriad of essential processes beyond protein synthesis.

Translation requires the specific attachment of amino acids to tRNAs by aminoacyl-tRNA synthetases (aaRSs) and the subsequent delivery of aminoacyl-tRNAs to the ribosome by elongation factor 1 alpha (EF-1α). Interactions between EF-1α and various aaRSs have been described in eukaryotes, but the role of these complexes remains unclear. To investigate possible interactions between EF-1α and other cellular components, a yeast two-hybrid screen was performed for the archaeon Methanothermobacter thermautotrophicus. EF-1α was found to form a stable complex with leucyl-tRNA synthetase (LeuRS; KD = 0.7 μM). Complex formation had little effect on EF-1α activity, but increased the kcat for Leu-tRNALeu synthesis ∼8-fold. In addition, EF-1α co-purified with the archaeal multi-synthetase complex (MSC) comprised of LeuRS, LysRS and ProRS, suggesting the existence of a larger aaRS:EF-1α complex in archaea. These interactions between EF-1α and the archaeal MSC contribute to translational fidelity both by enhancing the aminoacylation efficiencies of the three aaRSs in the complex and by coupling two stages of translation: aminoacylation of cognate tRNAs and their subsequent channeling to the ribosome.

Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multienzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA Pro aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K D of about 0.3-0.9 M, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the K m for tRNA Lys over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.To ensure that the correct amino acid is incorporated into the growing polypeptide chain during protein biosynthesis, the aminoacyl-tRNA synthetases (aaRSs) 2 must attach the correct amino acid to the corresponding tRNA molecule (1). This essential process, in turn, supplies the ribosome with the aminoacyl-tRNAs that are critical for the synthesis of proteins in all cells. The aaRSs can be divided into two groups, class I and II, based on the architecture of the catalytic domain (2-4). Class I aaRSs, which are normally monomeric, attach the aminoacyl group to the 2Ј-OH of the 3Ј terminal nucleotide of the tRNA, whereas class II aaRSs catalyze the addition to the 3Ј-OH of the tRNA and are usually dimeric. In bacteria, the aaRSs normally act alone as free-standing proteins, although in some instances their activities are enhanced by association with other translation factors (5, 6). In eukaryotic organisms, aaRSs are commonly found in multi-enzyme complexes within the cell that are believed to stabilize the interaction between tRNAs and synthetases, thereby increasing the efficiency of aminoacylation. These multi-enzyme complexes vary depending on the specific aaRSs and accessory proteins involved. For example, a complex forms between two synthetases and a non-synthetase protein in Saccharomyces cerevisiae (7), and there is also evidence for functional interactions between seryl-tRNA synthetase and a peroxisomal protein (8) as well as between tyrosyltRNA synthetase and a protein involved in the regulation of cell wall assembly in yeast (9). A complex of valyl-tRNA synthetase and human elongation factor-1H (10) as well as a larger complex consisting of nine aaRS activities...

Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible proteinprotein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA synthetase (MtArgRS) as an interacting partner of MtSerRS. Surface plasmon resonance confirmed stable complex formation, with a dissociation constant (K D ) of 250 nM. Formation of the MtSerRS⅐MtArgRS complex was further supported by the ability of GST-MtArgRS to co-purify MtSerRS and by coelution of the two enzymes during gel filtration chromatography. The MtSerRS⅐MtArgRS complex also contained tRNA Arg , consistent with the existence of a stable ribonucleoprotein complex active in aminoacylation. Steady-state kinetic analyses revealed that addition of MtArgRS to MtSerRS led to an almost 4-fold increase in the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that formation of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions. Aminoacyl-tRNA synthetases (aaRSs)2 catalyze the specific coupling of amino acids with their cognate tRNAs to produce aminoacyl-tRNAs (aa-tRNAs), which serve as starting materials for the biosynthesis of proteins. Aa-tRNA synthesis occurs in two steps: amino acid activation at the expense of ATP followed by the aminoacylation of tRNA (1). Although for most aaRSs the formation of aminoacyl-AMP does not require tRNA, cognate tRNA is necessary for amino acid activation by ArgRS, GlnRS, GluRS, and LysRS1 enzymes from many organisms (2). Based on structural features of their active sites, aaRSs can be divided into two classes, which comprise 10 members each (3). In addition, an unusual form of LysRS is found in class I (2), while class II also includes the noncanonical synthetases PylRS and SepRS (4).In all three domains of life, subsets of aaRSs have been shown to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSCs). These complexes are distinctive compared with other macromolecular protein complexes, because their components are enzymes that carry out similar catalytic reactions simultaneously, and only some aaRSs are involved (5). In eukaryotes, MSCs tend to be larger than those discovered in bacteria and archaea and also perform a wider range of functions that include both aminoacylation and noncanonical roles b...

Fungi secrete many different enzymes to deconstruct lignocellulosic biomass, including several families of hydrolases, oxidative enzymes, and many uncharacterized proteins. Here we describe the isolation, characterization, and primary sequence analysis of an extracellular aldonolactonase from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile). The lactonase is a 48-kDa glycoprotein with a broad pH optimum. The enzyme catalyzes the hydrolysis of glucono-␦-lactone and cellobiono-␦-lactone with an apparent second-order rate constant, k cat /K m , of ϳ1 ؋ 10 6 M ؊1 s ؊1 at pH 5.0 and 25°C but is unable to hydrolyze xylono-␥-lactone or arabino-␥-lactone. Sequence analyses of the lactonase show that it has distant homology to cis-carboxy-muconate lactonizing enzymes (CMLE) as well as 6-phosphogluconolactonases present in some bacteria. The M. thermophila genome contains two predicted extracellular lactonase genes, and expression of both genes is induced by the presence of pure cellulose. Homologues of the M. thermophila lactonase, which are also predicted to be extracellular, are present in nearly all known cellulolytic ascomycetes.Lignocellulosic biomass is an abundant renewable resource and a potential feedstock for the production of liquid fuels and other value-added products (25). The principal barriers to the production of lignocellulose-derived biofuels are the high costs of chemical pretreatment and enzymes for depolymerization (1). The thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile) very rapidly degrades cellulose and metabolizes powdered cellulose at nearly the same rate as glucose (4). The thermostability of the hydrolytic enzymes from this organism provides some practical advantages over the mesophilic fungus Hypocrea jecorina (synonym Trichoderma reesei), which has traditionally been used for the production of biomass-degrading enzymes. During growth on cellulosic substrates, M. thermophila secretes cellulases, hemicellulases (17), oxidative enzymes (6), and many proteins of unknown function.Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoprotein that is produced in large amounts by M. thermophila during growth on cellulose (6). CDH is produced by many cellulolytic fungi, although the biological function of the enzyme is still unclear (33). CDH oxidizes the reducing end of cellobiose and longer cellodextrins to the corresponding aldonolactones (Fig. 1). Sugar lactones have been shown to inhibit many different types of glycosyl hydrolases (9). For example, glucono-␦-lactone is a potent inhibitor of ␤-glucosidases (24). The product of CDH catalysis, cellobiono-␦-lactone, has been shown to be a strong inducer of cellulase in the filamentous fungus Hypocrea jecorina (synonym Trichoderma reesei) (15).However, the mechanism of cellulase induction and the metabolism of cellobiono-␦-lactone by fungi have not been studied in detail.Sugar lactones are unstable in aqueous solution and undergo hydrolysis to form the corresponding aldonic ...