Resilience and proteome response of Escherichia coli to high levels of isoleucine mistranslation.

Accurate pairing of amino acids and tRNAs is a prerequisite for faithful translation of genetic information during protein biosynthesis. Here we present the effects of proteome-wide mistranslation of isoleucine (Ile) by canonical valine (Val) or non-proteinogenic norvaline (Nva) in a genetically engineered Escherichia coli strain with an editing-defective isoleucyl-tRNA synthetase (IleRS). Editing-defective IleRS efficiently mischarges both Val and Nva to tRNAIle and impairs the translational accuracy of Ile decoding. When mistranslation was induced by the addition of Val or Nva to the growth medium, an Ile-to-Val or Ile-to-Nva substitution of up to 20 % was measured by high-resolution mass spectrometry. This mistranslation level impaired bacterial growth, promoted the SOS response and filamentation during stationary phase, caused global proteome dysregulation and upregulation of the cellular apparatus for maintaining proteostasis, including the major chaperones (GroES/EL, DnaK/DnaJ/GrpE and HtpG), the disaggregase ClpB and the proteases (Lon, HslV/HslU, ClpA, ClpS). The most important consequence of mistranslation appears to be non-specific protein aggregation, which is effectively counteracted by the disaggregase ClpB. Our data show that E. coli can sustain high isoleucine mistranslation levels and remain viable despite excessive protein aggregation and severely impaired translational fidelity. However, we show that inaccurate translation lowers bacterial resilience to heat stress and decreases bacterial survival at elevated temperatures.

Gly56 in the synthetic site of isoleucyl-tRNA synthetase confers specificity and maintains communication with the editing site.

Isoleucyl-tRNA synthetase (IleRS) links isoleucine to cognate tRNA via the Ile-AMP intermediate. Non-cognate valine is often mistakenly recognized as the IleRS substrate; therefore, to maintain the accuracy of translation, IleRS hydrolyzes Val-AMP within the synthetic site (pre-transfer editing). As this activity is not efficient enough, Val-tRNAIle is formed and hydrolyzed in the distant post-transfer editing site. A strictly conserved synthetic site residue Gly56 was previously shown to safeguard Ile-to-Val discrimination during aminoacyl (aa)-AMP formation. Here, we show that the Gly56Ala variant lost its specificity in pre-transfer editing, confirming that this residue ensures the selectivity of all synthetic site reactions. Moreover, we found that the Gly56Ala mutation affects IleRS interaction with aa-tRNA likely by disturbing tRNA-dependent communication between the two active sites.

Evolutionarily conserved cysteines in plant cytosolic seryl-tRNA synthetase are important for its resistance to oxidation.

We have previously identified a unique disulfide bond in the crystal structure of Arabidopsis cytosolic seryl-tRNA synthetase involving cysteines evolutionarily conserved in all green plants. Here, we discovered that both cysteines are important for protein stability, but with opposite effects, and that their microenvironment may promote disulfide bond formation in oxidizing conditions. The crystal structure of the C244S mutant exhibited higher rigidity and an extensive network of noncovalent interactions correlating with its higher thermal stability. The activity of the wild-type showed resistance to oxidation with H2 O2 , while the activities of cysteine-to-serine mutants were impaired, indicating that the disulfide link may enable the protein to function under oxidative stress conditions which can be beneficial for an efficient plant stress response.

Partitioning of the initial catalytic steps of leucyl-tRNA synthetase is driven by an active site peptide-plane flip.

To correctly aminoacylate tRNALeu, leucyl-tRNA synthetase (LeuRS) catalyzes three reactions: activation of leucine by ATP to form leucyl-adenylate (Leu-AMP), transfer of this amino acid to tRNALeu and post-transfer editing of any mischarged product. Although LeuRS has been well characterized biochemically, detailed structural information is currently only available for the latter two stages of catalysis. We have solved crystal structures for all enzymatic states of Neisseria gonorrhoeae LeuRS during Leu-AMP formation. These show a cycle of dramatic conformational changes, involving multiple domains, and correlate with an energetically unfavorable peptide-plane flip observed in the active site of the pre-transition state structure. Biochemical analyses, combined with mutant structural studies, reveal that this backbone distortion acts as a trigger, temporally compartmentalizing the first two catalytic steps. These results unveil the remarkable effect of this small structural alteration on the global dynamics and activity of the enzyme.

Uniform binding and negative catalysis at the origin of enzymes.

Enzymes are well known for their catalytic abilities, some even reaching "catalytic perfection" in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate-handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a "substrate-handle-binding-first" mechanism ("binding-first" for brevity) instead of "chemistry-first" and we are, therefore, left to wonder what the role of non-catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back-to-back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as "uniform binding" (parallel stabilization of all enzyme-bound states to the same degree). Indeed, we show that a uniform-binding proto-catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate-handle-binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction's course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of "binding-first" enzyme families like the aldolase superfamily.

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

The evolutionary history of the HUP domain.

Among the enzyme lineages that undoubtedly emerged prior to the last universal common ancestor is the so-called HUP, which includes Class I aminoacyl tRNA synthetases (AARSs) as well as enzymes mediating NAD, FAD, and CoA biosynthesis. Here, we provide a detailed analysis of HUP evolution, from emergence to structural and functional diversification. The HUP is a nucleotide binding domain that uniquely catalyzes adenylation via the release of pyrophosphate. In contrast to other ancient nucleotide binding domains with the αßα sandwich architecture, such as P-loop NTPases, the HUP's most conserved feature is not phosphate binding, but rather ribose binding by backbone interactions to the tips of ß1 and/or ß4. Indeed, the HUP exhibits unusual evolutionary plasticity and, while ribose binding is conserved, the location and mode of binding to the base and phosphate moieties of the nucleotide, and to the substrate(s) reacting with it, have diverged with time, foremost along the emergence of the AARSs. The HUP also beautifully demonstrates how a well-packed scaffold combined with evolvable surface elements promotes evolutionary innovation. Finally, we offer a scenario for the emergence of the HUP from a seed ßαß fragment, and suggest that despite an identical architecture, the HUP and the Rossmann represent independent emergences.

A pair of isoleucyl-tRNA synthetases in Bacilli fulfills complementary roles to keep fast translation and provide antibiotic resistance.

Isoleucyl-tRNA synthetase (IleRS) is an essential enzyme that covalently couples isoleucine to the corresponding tRNA. Bacterial IleRSs group in two clades, ileS1 and ileS2, the latter bringing resistance to the natural antibiotic mupirocin. Generally, bacteria rely on either ileS1 or ileS2 as a standalone housekeeping gene. However, we have found an exception by noticing that Bacillus species with genomic ileS2 consistently also keep ileS1, which appears mandatory in the family Bacillaceae. Taking Priestia (Bacillus) megaterium as a model organism, we showed that PmIleRS1 is constitutively expressed, while PmIleRS2 is stress-induced. Both enzymes share the same level of the aminoacylation accuracy. Yet, PmIleRS1 exhibited a two-fold faster aminoacylation turnover (kcat ) than PmIleRS2 and permitted a notably faster cell-free translation. At the same time, PmIleRS2 displayed a 104 -fold increase in its Ki for mupirocin, arguing that the aminoacylation turnover in IleRS2 could have been traded-off for antibiotic resistance. As expected, a P. megaterium strain deleted for ileS2 was mupirocin-sensitive. Interestingly, an attempt to construct a mupirocin-resistant strain lacking ileS1, a solution not found among species of the family Bacillaceae in nature, led to a viable but compromised strain. Our data suggest that PmIleRS1 is kept to promote fast translation, whereas PmIleRS2 is maintained to provide antibiotic resistance when needed. This is consistent with an emerging picture in which fast-growing organisms predominantly use IleRS1 for competitive survival.

Polyamines Mediate Folding of Primordial Hyperacidic Helical Proteins.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

How evolution shapes enzyme selectivity - lessons from aminoacyl-tRNA synthetases and other amino acid utilizing enzymes.

Aminoacyl-tRNA synthetases (AARSs) charge tRNA with their cognate amino acids. Many other enzymes use amino acids as substrates, yet discrimination against noncognate amino acids that threaten the accuracy of protein translation is a hallmark of AARSs. Comparing AARSs to these other enzymes allowed us to recognize patterns in molecular recognition and strategies used by evolution for exercising selectivity. Overall, AARSs are 2-3 orders of magnitude more selective than most other amino acid utilizing enzymes. AARSs also reveal the physicochemical limits of molecular discrimination. For example, amino acids smaller by a single methyl moiety present a discrimination ceiling of ~200, while larger ones can be discriminated by up to 105 -fold. In contrast, substrates larger by a hydroxyl group challenge AARS selectivity, due to promiscuous H-bonding with polar active site groups. This 'hydroxyl paradox' is resolved by editing. Indeed, when the physicochemical discrimination limits are reached, post-transfer editing - hydrolysis of tRNAs charged with noncognate amino acids, evolved. The editing site often selectively recognizes the edited noncognate substrate using the very same feature that the synthetic site could not efficiently discriminate against. Finally, the comparison to other enzymes also reveals that the selectivity of AARSs is an explicitly evolved trait, showing some clear examples of how selection acted not only to optimize catalytic efficiency with the target substrate, but also to abolish activity with noncognate threat substrates ('negative selection').

Mechanism of discrimination of isoleucyl-tRNA synthetase against nonproteinogenic α-aminobutyrate and its fluorinated analogues.

Isoleucyl-tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200-fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α-aminobutyrate (Abu) is activated 30-fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM . To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary-related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu-tRNAIle with a rate of 40 s-1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. ENZYMES: Isoleucyl-tRNA synthetase (EC 6.1.1.5), leucyl-tRNA synthetase (EC 6.1.1.4), valyl-tRNA synthetase (EC 6.1.1.9).

On the Mechanism and Origin of Isoleucyl-tRNA Synthetase Editing against Norvaline.

Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) in their propensity to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), Nva and Val are activated and transferred to tRNA at similar rates. The efficiency of the synthetic site in pre-transfer editing of Nva and Val also appears to be similar. Post-transfer editing was, however, more rapid with Nva and consequently IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS editing being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary-related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. We thus hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins.

Arabidopsis seryl-tRNA synthetase: the first crystal structure and novel protein interactor of plant aminoacyl-tRNA synthetase.

The rules of the genetic code are established by aminoacyl-tRNA synthetases (aaRSs) enzymes, which covalently link tRNA with the cognate amino acid. Many aaRSs are involved in diverse cellular processes beyond translation, acting alone, or in complex with other proteins. However, studies of aaRS noncanonical assembly and functions in plants are scarce, as are structural studies of plant aaRSs. Here, we have solved the crystal structure of Arabidopsis thaliana cytosolic seryl-tRNA synthetase (SerRS), which is the first crystallographic structure of a plant aaRS. Arabidopsis SerRS displays structural features typical of canonical SerRSs, except for a unique intrasubunit disulfide bridge. In a yeast two-hybrid screen, we identified BEN1, a protein involved in the metabolism of plant brassinosteroid hormones, as a protein interactor of Arabidopsis SerRS. The SerRS:BEN1 complex is one of the first protein complexes of plant aaRSs discovered so far, and is a rare example of an aaRS interacting with an enzyme involved in primary or secondary metabolism. To pinpoint regions responsible for this interaction, we created truncated variants of SerRS and BEN1, and identified that the interaction interface involves the SerRS globular catalytic domain and the N-terminal extension of BEN1 protein. BEN1 does not have a strong impact on SerRS aminoacylation activity, indicating that the primary function of the complex is not the modification of SerRS canonical activity. Perhaps SerRS performs as yet unknown noncanonical functions mediated by BEN1. These findings indicate that - via SerRS and BEN1 - a link exists between the protein translation and steroid metabolic pathways of the plant cell. DATABASE: Structural data are available in the PDB under the accession number PDB ID 6GIR.

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase.

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNALeu differed by about 104-fold. We further established the critical role for the A76 3'-OH group of the tRNALeu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNALeu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.

An archaeal aminoacyl-tRNA synthetase complex for improved substrate quality control.

Aminoacyl-tRNA synthetases (aaRSs) decode genetic information by coupling tRNAs with cognate amino acids. In the archaeon Methanothermobacter thermautotrophicus arginyl- and seryl-tRNA synthetase (ArgRS and SerRS, respectively) form a complex which enhances serylation and facilitates tRNASer recycling through its association with the ribosome. Yet, the way by which complex formation participates in Arg-tRNAArg synthesis is still unresolved. Here we utilized pull down and surface plasmon resonance experiments with truncated ArgRS variants to demonstrate that ArgRS uses its N-terminal domain to establish analogous interactions with both SerRS and cognate tRNAArg, providing a rationale for the lack of detectable SerRS•[ArgRS•tRNAArg] complex. In contrast, stable ternary ArgRS•[SerRS•tRNASer] complex was easily detected supporting the model wherein ArgRS operates in serylation by modulating SerRS affinity toward tRNASer. We also found that the interaction with SerRS suppresses arginylation of unmodified tRNAArg by ArgRS, which, by itself, does not discriminate against tRNAArg substrates lacking posttranscriptional modifications. Hence, there is a fundamentally different participation of the protein partners in Arg-tRNA and Ser-tRNA synthesis. Propensity of the ArgRS•SerRS complex to exclude unmodified tRNAs from translation leads to an attractive hypothesis that SerRS•ArgRS complex might act in vivo as a safeguarding switch that improves translation accuracy.

Discovery and Investigation of Natural Editing Function against Artificial Amino Acids in Protein Translation.

Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) from Escherichia coli to investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNAIle in the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNAIle from IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids.

Synthetic and editing reactions of aminoacyl-tRNA synthetases using cognate and non-cognate amino acid substrates.

The covalent coupling of cognate amino acid-tRNA pairs by corresponding aminoacyl-tRNA synthetases (aaRS) defines the genetic code and provides aminoacylated tRNAs for ribosomal protein synthesis. Besides the cognate substrate, some non-cognate amino acids may also compete for tRNA aminoacylation. However, their participation in protein synthesis is generally prevented by an aaRS proofreading activity located in the synthetic site and in a separate editing domain. These mechanisms, coupled with the ability of certain aaRSs to discriminate well against non-cognate amino acids in the synthetic reaction alone, define the accuracy of the aminoacylation reaction. aaRS quality control may also act as a gatekeeper for the standard genetic code and prevents infiltration by natural amino acids that are not normally coded for protein biosynthesis. This latter finding has reinforced interest in understanding the principles that govern discrimination against a range of potential non-cognate amino acids. This paper presents an overview of the kinetic assays that have been established for monitoring synthetic and editing reactions with cognate and non-cognate amino acid substrates. Taking into account the peculiarities of non-cognate reactions, the specific controls needed and the dedicated experimental designs are discussed in detail. Kinetic partitioning within the synthetic and editing sites controls the balance between editing and aminoacylation. We describe in detail steady-state and single-turnover approaches for the analysis of synthetic and editing reactions, which ultimately enable mechanisms of amino acid discrimination to be determined.

Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications.

The genetic code is virtually universal in biology and was likely established before the advent of cellular life. The extent to which mistranslation occurs is poorly understood and presents a fundamental question in basic research and production of recombinant proteins. Here we used shotgun proteomics combined with unbiased protein modification analysis to quantitatively analyze in vivo mistranslation in an E. coli strain with a defect in the editing mechanism of leucyl-tRNA synthetase. We detected the misincorporation of a non-proteinogenic amino acid norvaline on 10% of all measured leucine residues under microaerobic conditions and revealed preferential deployment of a tRNA(Leu)(CAG) isoacceptor during norvaline misincorporation. The strain with the norvalylated proteome demonstrated a substantial reduction in cell fitness under both prolonged aerobic and microaerobic cultivation. Unlike norvaline, isoleucine did not substitute for leucine even under harsh error-prone conditions. Our study introduces shotgun proteomics as a powerful tool in quantitative analysis of mistranslation.

Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing.

Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 10(3)-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability.

The origin of specificity and insight into recognition between an aminoacyl carrier protein and its partner ligase.

Acyl carrier proteins (ACPs) are among the most promiscuous proteins in terms of protein-protein interactions and it is quite puzzling how ACPs select the correct partner between many possible upstream and downstream binding proteins. To address this question, we performed molecular dynamics simulations on dimeric Bradyrhizobium japonicum Gly:CP ligase 1 to inspect the origin of its selectivity towards the three types of carrier proteins, namely holoCP, apoCP, and holoCP-Gly, which only differ in the attached prosthetic group. In line with experiments, MM-GBSA analysis revealed that the ligase preferentially binds the holoCP form to both subunits with the binding free energies of -20.7 and -19.1 kcal mol(-1), while the apoCP form, without the prosthetic group, is also recognized, but the binding values of -9.2 and -3.6 kcal mol(-1) suggest that there is no competition for the ligase binding as long as the holoCP is present. After the prosthetic group becomes glycylated, the holoCP-Gly dissociates from the ligase, as supported by its endergonic binding free energies of 2.9 and 20.9 kcal mol(-1). Our results indicate that these affinity differences are influenced by three aspects: the form of the prosthetic group and the specific non-polar hydrophobic interactions, as well as charge complementarity dominantly manifested through Arg220-Glu53 ion pair within the binding region among proteins. A careful examination of the bonding patterns within the ligase active site elucidated the interactions with Arg258, Asp215 and Tyr132 as being predominant in stabilizing the prosthetic group, which are significantly diminished upon glycation, thus promoting complex dissociation.