seryl-tRNA Synthetase Research Articles

Identification of a novel candidate gene for rolled leaf in rice

A semi-narrow and adaxially rolled leaf mutant, rl15(t), was induced from Korean japonica rice cultivar Ilpum by chemical mutagenesis using ethyl methanesulfonate. We characterized the mutant and identified the novel gene causing the mutant phenotype. Cytological analysis of mutant leaves indicated that the adaxial leaf-rolling phenotype is due to the reduced size and number of bulliform cells in the mutant. Genetic analysis showed that the rolled leaf trait is controlled by a single recessive gene, designated rl15(t). Using an F2 mapping population generated from a cross between Milyang23 and the mutant, we mapped the candidate region to a 174 kb interval on the long arm of chromosome 1 near the centromeric region. Through whole genome sequencing in bulk and MutMap analysis, we identified the causal SNP within the candidate region. The results of RT-PCR analysis indicated that a splicing error occurred due to a base change from G to A at the beginning of the fifth intron of LOC_Os01g37837, which encodes a putative seryl-tRNA synthetase, resulting in the mutant phenotype. Further study of the rl15(t) gene will facilitate analysis of leaf architecture and morphogenesis in rice plants.

Splicing Defect in Mitochondrial Seryl-tRNA Synthetase Gene Causes Progressive Spastic Paresis Instead of HUPRA Syndrome

Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function.

Exclusive cytosolic localization and broad tRNASer specificity of Arabidopsis thaliana seryl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) decipher the genetic code, covalently linking amino acids to cognate tRNAs, thus preparing substrates for the process of translation. Although aaRSs funtion primarily in translation and are localized in cytosol, mitochondria and chloroplasts there are many reports on their additional functions and subcellular destinations beyond translation. However, data on plant aaRSs are scarce. Initial analysis of amino acid sequence of Arabidopsis thaliana seryl-tRNA synthetase (SerRS) suggested that protein contains putative nuclear localization signals. GFP-localization experiments in transiently transformed epidermal onion cells and Arabidopsis protoplasts gave ambiguous results because in some cells SerRS appeared to be dually localized to both cytosol and nucleus. However, data obtained on transgenic lines expressing SerRS-TAP and GFP-SerRS revealed exclusive cytosolic location of SerRS. Subcellular distribution of SerRS did not change during stress. Cytosolic Arabidopsis SerRS was expressed and purified. The enzyme efficiently aminoacylated eukaryotic and bacterial tRNAsSer, that are structurally very different. Given the fact that the same behavior was previously shown for monocot maize SerRS, it seems that plant SerRSs exhibit unusually broad tRNASer specificity, unlike SerRSs from other organisms. Possible functional implications of this unique characteristic of plant SerRSs are discussed.

Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase

The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different.

Seryl-tRNA Synthetases in Translation and Beyond

Seryl-tRNA Synthetases in Translation and Beyond

SerRS-tRNASec complex structures reveal mechanism of the first step in selenocysteine biosynthesis.

Selenocysteine (Sec) is found in the catalytic centers of many selenoproteins and plays important roles in living organisms. Malfunctions of selenoproteins lead to various human disorders including cancer. Known as the 21st amino acid, the biosynthesis of Sec involves unusual pathways consisting of several stages. While the later stages of the pathways are well elucidated, the molecular basis of the first stage—the serylation of Sec-specific tRNA (tRNASec) catalyzed by seryl-tRNA synthetase (SerRS)—is unclear. Here we present two cocrystal structures of human SerRS bound with tRNASec in different stoichiometry and confirm the formation of both complexes in solution by various characterization techniques. We discovered that the enzyme mainly recognizes the backbone of the long variable arm of tRNASec with few base-specific contacts. The N-terminal coiled-coil region works like a long-range lever to precisely direct tRNA 3′ end to the other protein subunit for aminoacylation in a conformation-dependent manner. Restraints of the flexibility of the coiled-coil greatly reduce serylation efficiencies. Lastly, modeling studies suggest that the local differences present in the D- and T-regions as well as the characteristic U20:G19:C56 base triple in tRNASec may allow SerRS to distinguish tRNASec from closely related tRNASer substrate.

Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation

Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNA(Pro) and Cys-tRNA(Pro), respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed "ProXp-ST1" and "ProXp-ST2," respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.

Ancient horizontal gene transfer and the last common ancestors.

BackgroundThe genomic history of prokaryotic organismal lineages is marked by extensive horizontal gene transfer (HGT) between groups of organisms at all taxonomic levels. These HGT events have played an essential role in the origin and distribution of biological innovations. Analyses of ancient gene families show that HGT existed in the distant past, even at the time of the organismal last universal common ancestor (LUCA). Most gene transfers originated in lineages that have since gone extinct. Therefore, one cannot assume that the last common ancestors of each gene were all present in the same cell representing the cellular ancestor of all extant life.ResultsOrganisms existing as part of a diverse ecosystem at the time of LUCA likely shared genetic material between lineages. If these other lineages persisted for some time, HGT with the descendants of LUCA could have continued into the bacterial and archaeal lineages. Phylogenetic analyses of aminoacyl-tRNA synthetase protein families support the hypothesis that the molecular common ancestors of the most ancient gene families did not all coincide in space and time. This is most apparent in the evolutionary histories of seryl-tRNA synthetase and threonyl-tRNA synthetase protein families, each containing highly divergent “rare” forms, as well as the sparse phylogenetic distributions of pyrrolysyl-tRNA synthetase, and the bacterial heterodimeric form of glycyl-tRNA synthetase. These topologies and phyletic distributions are consistent with horizontal transfers from ancient, likely extinct branches of the tree of life.ConclusionsOf all the organisms that may have existed at the time of LUCA, by definition only one lineage is survived by known progeny; however, this lineage retains a genomic record of heterogeneous genetic origins. The evolutionary histories of aminoacyl-tRNA synthetases (aaRS) are especially informative in detecting this signal, as they perform primordial biological functions, have undergone several ancient HGT events, and contain many sites with low substitution rates allowing deep phylogenetic reconstruction. We conclude that some aaRS families contain groups that diverge before LUCA. We propose that these ancient gene variants be described by the term “hypnologs”, reflecting their ancient, reticulate origin from a time in life history that has been all but erased”.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0350-0) contains supplementary material, which is available to authorized users.

Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the reaction center. Synchronously, Arg366 of the β sheet at the base holds the syn oxygen of the attacking carboxylic group so that the attack by the anti oxygen is feasible. This residue also contributes to the reduction of the unfavorable electrostatic potential at the reaction center. Present simulation clearly shows the catalytic role of the residues at reaction center. A precise and stable geometry of hydrogen bonded network develops within the active site, which is essential for the development of an optimum transition state geometry. All loops move away from the platform of active site in the open or adenylate bound state and the network of hydrogen bond disappears. The serine binding site is most rigid among all three subsites. The Ser is held here in a highly organized geometry bound by Zn(2+) and Cys residues. Present simulation further suggests that the helix-turn-helix motif connecting the monomers might have important role in coordinating the functional dynamics of the two active sites. The N-terminal domain is involved in long-range electrostatic interaction and specific hydrogen bond interaction (both direct and water mediated) with tRNA. Overall conformational fluctuation is less in the N terminal compared to the catalytic domain due to the presence of a motif 2 loop, loop f, and serine ordering loop, which change conformation in the later domain during the reaction cycle. The dynamic perspective of the active site of (mk)SerRS with the mobile loop acting as the gate and dynamically silent β sheets performing as the base has similarity with the perception of the active site in various other enzymes.

Microfluidic analysis of serine levels using seryl-tRNA synthetase coupled with spectrophotometric detection.

The measurement of amino acid content is useful for the diagnosis of several types of diseases, including cancer and diabetes. In this study, a microfluidic method for the analysis of serine using enzymatic reactions coupled with spectrophotometric detection was developed. The assay system has some advantages in the analytical field, such as the ability to detect small amounts of analyte and reaction solution and a rapid and efficient reaction. For the specific detection of serine, seryl-tRNA synthetase was coupled with the generation of hydrogen peroxide, which was then detected by the Trinder reagent spectrophotometric method. Seryl- and other aminoacyl-tRNA synthetases are involved in the biosynthesis of peptides and proteins in the human body and should allow precise recognition of the corresponding amino acids. This approach provided selective quantitation of up to 250 μM serine in 100 mM Tris-HCl buffer (pH 8.0) in a semiautomatic system.

Structure of the Entire Stalk Region of the Dynein Motor Domain

Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, as well as the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA+ modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA+ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~15nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state and with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamics simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil and imply the manner of change between high-affinity state and low-affinity state of MTBD.

The RimL transacetylase provides resistance to translation inhibitor microcin C.

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.

TRNA synthetase counteracts c-Myc to develop functional vasculature.

Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of 'Yin-Yang' transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.DOI: http://dx.doi.org/10.7554/eLife.02349.001.

Analysis of Debye Waller factor and molecular dynamics simulation of Seryl tRNA synthetase

Analysis of Debye Waller factor and molecular dynamics simulation of Seryl tRNA synthetase

Functional Epigenetic Analysis of Prostate Carcinoma: A Role for Seryl-tRNA Synthetase?

Transcriptional silencing, as a result of aberrant promoter hypermethylation, is a common mechanism through which genes in cancer cells become inactive. Functional epigenetic screens using demethylating agents to reexpress transcriptional silenced genes may identify such inactivated genes for needing further evaluation. We aimed to identify genes so far not known to be inactivated by promoter hypermethylation in prostate cancer. DU-145 and LNCaP cells were treated with the DNMT inhibitor zebularine. Expression changes of total RNA from treated and untreated cells were compared using an RNA expression microarray. Genes upregulated more than 2-fold were evaluated by RT-qPCR in 50 cases of paired normal and tumor tissues of prostate cancer patients. SARS was found to be downregulated in prostate cancer in 42/50 cases (84%). In addition, GADD45A and SPRY4 showed a remarkable diminished expression (88% and 74%, resp.). The gold standard for promoter hypermethylation-inactivated genes in prostate cancer (GSTP1) was repressed in 90% of our patient samples. ROC analyses reported statistically significant AUC curves in SARS, GADD45A, and GSTP1 and positive Spearman correlations were found between these genes. SARS was discovered to be a novel gene that is repressed in prostate cancer and could therefore be recommended for its involvement in prostate carcinogenesis.

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617-650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617-650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia-c and IIa-c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes--(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.

Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.

A single amino acid substitution affects the substrate specificity of the seryl-tRNA synthetase homologue.

Recently described and characterized Bradyrhizobium japonicum glycine:[carrier protein] ligase 1 (Bj Gly:CP ligase 1), a homologue of methanogenic type seryl-tRNA synthetase (SerRS) is an intriguing enzyme whose physiological role is not yet known. While aminoacyl-tRNA synthetases supply ribosome with amino acids for protein biosynthesis, this homologue transfers the activated amino acid to a specific carrier protein. Despite remarkable structural similarity between the Bj Gly:CP ligase 1 and the catalytic core domain of methanogenic type SerRS, the ligase displays altered and relaxed substrate specificity. In contrast to methanogenic SerRS which exclusively activates serine, the Bj Gly:CP ligase 1 predominantly activates glycine. Besides, it shows low activity in the presence of alanine, but it is incapable of activating serine. The detailed computational study aiming to address this unexpected substrate specificity toward the small aliphatic amino acids revealed the A281G Bj Gly:CP ligase 1 mutant as the most promising candidate with reconstituted catalytic activity toward the larger substrates. The A281G mutation is predicted to increase the active site volume, allowing alanine and serine to establish important hydrogen bonds within the active site, and to adopt an optimal orientation for the reaction. The results were tested by the site-directed mutagenesis experiments coupled with in vitro kinetic assays. It was found that the A281G substitution greatly affects the enzyme specificity and allows efficient activation of both polar and small aliphatic amino acids (serine, glycine and alanine), confirming predictions and conclusions based on molecular dynamics simulations.

21番目のアミノ酸, セレノシステイン合成の分子機構

Selenocysteine (Sec) is the 21st amino acid that is incorporated into proteins translationally. The selenocysteine tRNA (tRNASec) is first ligated with serine by seryl-tRNA synthetase, and the Ser moiety is converted to Sec in a tRNA-dependent manner. In Bacteria, the selenocysteine synthase SelA converts Ser to Sec directly, whereas in Eukaryotes and Archaea, phosphorylation of the Ser moiety is required prior to the Sec conversion. In this review, we describe the crystal structures of SelA, bacterial tRNASec, and the SelA•tRNASec complex. Based on the crystal structures and mutational analyses, the substrate discrimination and reaction mechanisms are discussed.