Characterizing the amino acid activation center of the naturally editing-deficient aminoacyl-tRNA synthetase PheRS in Mycoplasma mobile.

To ensure that correct amino acids are incorporated during protein synthesis, aminoacyl-tRNA synthetases (aaRSs) use proofreading mechanisms collectively referred to as editing. Although editing is important for viability, editing-deficient aaRSs have been identified in host-dependent organisms. In Mycoplasma mobile, editing-deficient PheRS and LeuRS have been identified. We characterized the amino acid activation site of MmPheRS and identified a previously unknown hyperaccurate mutation, L287F. Additionally, we report that m-Tyr, an oxidation byproduct of Phe which is toxic to editing-deficient cells, is poorly discriminated by MmPheRS activation and is not subjected to editing. Furthermore, expressing MmPheRS and the hyperaccurate variants renders Escherichia coli susceptible to m-Tyr stress, indicating that active site discrimination is insufficient in tolerating excess m-Tyr.

Chained Structure of Dimeric F1-like ATPase in Mycoplasma mobile Gliding Machinery.

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called "chains" that are each composed of 17 repeating protein units called "particles." These proteins include homologs of the catalytic α and ß subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOB1660 (α-subunit homolog), -1670 (ß-subunit homolog), -1630, -1620, and -4530, and showed ATP hydrolyzing activity. The two-dimensional (2D) structure, with dimensions of 35 and 26 nm, showed a dimer of hexameric ring approximately 12 nm in diameter, resembling F1-ATPase catalytic (αß)3. We isolated the F1-like ATPase unit, which is composed of MMOB1660, -1670, and -1630. Furthermore, we isolated the chain and analyzed the three-dimensional (3D) structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31-nm intervals. An atomic model of F1-ATPase catalytic (αß)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCE F1Fo-ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

Crystal Structures and Catalytic Mechanism of l-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of ß-Amino Acids.

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about ß-AADHs that enable the synthesis of ß-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of ß-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic ß-amino acids without an enantioselectivity trade-off. Two ß-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of ß-AADHs for the asymmetric synthesis of ß-amino acids.

Refined Mechanism of Mycoplasma mobile Gliding Based on Structure, ATPase Activity, and Sialic Acid Binding of Machinery.

Mycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal "jellyfish"-like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal "tentacle"-like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the α- and ß-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and showed that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding.IMPORTANCE The genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human "walking pneumonia," is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms, always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.

Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

In silico Analysis of Different Signal Peptides for Secretory Production of Arginine Deiminase in Escherichia coli.

BACKGROUND: Secretory production of recombinant protein in bacterial hosts fulfills several advantages. Selecting an appropriate secretory signal peptide is a critical step in secretory production of different protein. Several patents report the usage of signal peptides for secretory production of recombinant proteins in E. coli. In silico identification of suitable signal peptides is a reliable and cost-effective alternative to experimental approaches. OBJECTIVE: This study was aimed to predict best signal peptides for the secretory production of recombinant arginine deiminase in E. coli. METHODS: In this study, 30 different signal peptide sequences were retrieved from database. The signal peptide probability, location of cleavage sites, and n, h and c regions were predicted by SignalP 4.1 and Phobius servers. After purging the 30 predicted secretory signal peptides, TorT, bla, NrfA, TolB, PapC, PldA, Lpp were removed. Several physicochemical properties of the remaining potential SPs were determined by ProtParam, PROSO II, and SOLpro servers for theoretically selecting the best candidates. RESULTS AND CONCLUSION: Based on physicochemical properties, the signal peptides of OmpC, OmpF, and DsbA were identified respectively as the promising candidates for efficient secretory production of arginine deiminase in E. coli. Although the computational approach has established itself as a basis of modern biotechnology, the experimental study is necessary to validate its results. The criteria used in this study could be applied to other targets for recombination processes.

Sialidase and N-acetylneuraminate catabolism in nutrition of Mycoplasma alligatoris.

The contribution of N-acetylneuraminate scavenging to the nutrition of Mycoplasma alligatoris was examined. The wild-type grew substantially faster (P<0.01) than the mutant strains that were unable either to liberate (extracellular NanI- mutants) or to catabolize (NanA- mutants) N-acetylneuraminate from glycoconjugates in minimal SP-4 medium supplemented only with serum, but the growth of sialidase-negative mutants could not be restored to wild-type rate simply by adding unconjugated sialic acid to the culture medium. In 1 : 1 growth competition assays the wild-type was recovered in >99-fold excess of a sialidase-negative mutant after co-culture on pulmonary fibroblasts in serum-free RPMI 1640 medium, even with supplemental glucose. The advantage of nutrient scavenging via this mechanism in a complex glycan-rich environment may help to balance the expected selective disadvantage conferred by the pathogenic effects of mycoplasmal sialidase in an infected host.

Arginine Deiminase: Current Understanding and Applications.

BACKGROUND: Arginine deiminase (ADI), an arginine catabolizing enzyme, is considered as an anti-tumor agent for the treatment of arginine auxotrophic cancers. However, some obstacles limit its clinical applications. OBJECTIVE: This review will summarize the clinical applications of ADI, from a brief history to its limitations, and will discuss the different ways to deal with the clinical limitations. METHOD: The structure analysis, cloning, expression, protein engineering and applications of arginine deiminase enzyme have been explained in this review. CONCLUSION: Recent patents on ADI are related to ADI engineering to increase its efficacy for clinical application. The intracellular delivery of ADI and combination therapy seem to be the future strategies in the treatment of arginine auxotrophic cancers. Applying ADIs with optimum features from different sources and or ADI engineering, are promising strategies to improve the clinical application of ADI.

Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens.

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5-7. Here we describe a group of RNR proteins in Mollicutes-including Mycoplasma pathogens-that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR-some of which are developing resistance to antibiotics-are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

Arginine Deiminase Enzyme Evolving as a Potential Antitumor Agent.

Some melanomas and hepatocellular carcinomas have been shown to be auxotrophic for arginine. Arginine deiminase (ADI), an arginine degrading enzyme isolated from Mycoplasma, can inhibit the growth of these tumors. It is a catabolizing enzyme which catabolizes arginine to Citrulline. Tumor cells do not express an enzyme called arginosuccinate synthetase (ASS) and hence, these cells become auxotrophic for arginine. It is found that ADI is specific for arginine and did not degrade other amino acid. This review covers various aspects of ADIs like origin, properties and chemical modifications for better antitumor activity.

Engineering the GH1 ß-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose.

The activity of the GH1 ß-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-ß-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides.

Medium Optimization for Recombinant Soluble Arginine Deiminase Expression in Escherichia coli Using Response Surface Methodology.

OBJECTIVE: Optimization of the medium for recombinant arginine deiminase production in E. coli was performed using response surface methodology. This is the first study of optimization of recombinant arginine deiminase production in E. coli by the use of response surface methodology. METHODS: A Mycoplasm arginine deiminase gene was computationally optimized and inserted into pET-3a (+) expression vector. The synthetic pET3a-arginine deiminase construct was cloned and overexpressed in E. coli. The effect of glucose, NH4Cl and MgSO4.7H2O concentrations on the expression of intracellular soluble arginine deiminase was investigated using central composite design (CCD). RESULTS: The maximum arginine deiminase activity (U/mL) was obtained in optimal concentrations g/L of glucose, 6.6; NH4Cl, 1.81; MgSO4.7H2O, 0.94; KH2PO4, 3.0; Na2HPO4, 6.78; NaCl, 0.5; CaCl2, 0.1 mL/L (1M), which was about 6.6 fold higher than that in M9 standard medium. CONCLUSION: The obtained result can be utilized for large-scale production of this enzyme and related recombinant protein.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module.

A conserved structural module following the KMSKS catalytic loop exhibits α-α-ß-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the ß-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS.

Synthesis of Mycoplasma arginine deiminase in E. coli using stress-responsive proteins.

We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

A small GTPase­like protein fragment of Mycoplasma promotes tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

The Mycoplasma genus comprises a group of microbes that cause persistent infection in humans and its role in promoting tumor development has long been a concern. Although mixtures of components isolated from Mycoplasma have been shown to activate host Rho family small GTPases and Stat3, no individual factor with this activity has been reported. In the current study, a conserved small GTPase-like protein fragment (SGLP) from Mycoplasma pulmonis chromosome partition protein, Smc, was identified as a virulence factor. SGLP was observed to interact with Rac1 and Stat3. The wild­type (wt) SGLP, which contains a WxxxE motif, induced activation of Rac1 and phosphorylation of Stat3 at the tyrosine­705 residue, while the SGLP mutant containing a mutation from WxxxE to AxxxA did not exert the same effects. Moreover, SGLP­induced Stat3 phosphorylation was observed to be dependent upon Rac1 activity. Furthermore, wt SGLP was observed to promote cell migration and increase bromodeoxyuridine incorporation in HeLa cells and the SGLP mutant did not elicit these effects in HeLa cells. In conclusion, the current observations suggest that SGLP is an important virulence factor of Mycoplasma, which contributes to tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

Structure-function features of a Mycoplasma glycolipid synthase derived from structural data integration, molecular simulations, and mutational analysis.

Glycoglycerolipids are structural components of mycoplasma membranes with a fundamental role in membrane properties and stability. Their biosynthesis is mediated by glycosyltransferases (GT) that catalyze the transfer of glycosyl units from a sugar nucleotide donor to diacylglycerol. The essential function of glycolipid synthases in mycoplasma viability, and the absence of glycoglycerolipids in animal host cells make these GT enzymes a target for drug discovery by designing specific inhibitors. However, rational drug design has been hampered by the lack of structural information for any mycoplasma GT. Most of the annotated GTs in pathogenic mycoplasmas belong to family GT2. We had previously shown that MG517 in Mycoplasma genitalium is a GT-A family GT2 membrane-associated glycolipid synthase. We present here a series of structural models of MG517 obtained by homology modeling following a multiple-template approach. The models have been validated by mutational analysis and refined by long scale molecular dynamics simulations. Based on the models, key structure-function relationships have been identified: The N-terminal GT domain has a GT-A topology that includes a non-conserved variable region involved in acceptor substrate binding. Glu193 is proposed as the catalytic base in the GT mechanism, and Asp40, Tyr126, Tyr169, Ile170 and Tyr218 define the substrates binding site. Mutation Y169F increases the enzyme activity and significantly alters the processivity (or sequential transferase activity) of the enzyme. This is the first structural model of a GT-A glycoglycerolipid synthase and provides preliminary insights into structure and function relationships in this family of enzymes.

Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

Decoding system for the AUA codon by tRNAIle with the UAU anticodon in Mycoplasma mobile.

Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNA(Ile2) bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNA(Ile)-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNA(Ile2) has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNA(Ile2)(UAU). In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNA(Ile2) cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNA(Ile2)(UAU) specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system.