RESUMO
We report a 2.0 Å structure of the CAE31940 protein, a proteobacterial NMT1/THI5-like domain-containing protein. We also discuss the primary and tertiary structure similarity with its homologs. The highly conserved FGGXMP motif was identified in CAE31940, which corresponds to the GCCCX motif located in the vicinity of the active center characteristic for THi5-like proteins found in yeast. This suggests that the FGGXMP motif may be a unique hallmark of proteobacterial NMT1/THI5-like proteins.
Assuntos
Proteínas de Bactérias/química , Bordetella bronchiseptica , Evolução Molecular , Sequência de Aminoácidos , Cristalografia por Raios X , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na(+) efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all. Structural analysis of calcium ion binding sites found at the interface between TM0543 dimer subunits suggests that protein may be involved in ion-transporting activity. A detailed analysis of the protein sequence and structure is presented and discussed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Thermotoga maritima/química , Sítios de Ligação , Cristalografia por Raios X , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The explosion of the size of the universe of known protein sequences has stimulated two complementary approaches to structural mapping of these sequences: theoretical structure prediction and experimental determination by structural genomics (SG). In this work, we assess the accuracy of structure prediction by two automated template-based structure prediction metaservers (genesilico.pl and bioinfo.pl) by measuring the structural similarity of the predicted models to corresponding experimental models determined a posteriori. Of 199 targets chosen from SG programs, the metaservers predicted the structures of about a fourth of them "correctly." (In this case, "correct" was defined as placing more than 70 % of the alpha carbon atoms in the model within 2 Å of the experimentally determined positions.) Almost all of the targets that could be modeled to this accuracy were those with an available template in the Protein Data Bank (PDB) with more than 25 % sequence identity. The majority of those SG targets with lower sequence identity to structures in the PDB were not predicted by the metaservers with this accuracy. We also compared metaserver results to CASP8 results, finding that the models obtained by participants in the CASP competition were significantly better than those produced by the metaservers.
Assuntos
Biologia Computacional/métodos , Genômica/métodos , Conformação Proteica , Análise de Sequência de Proteína/métodos , Software , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteômica/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Alinhamento de Sequência , Homologia de SequênciaRESUMO
Isochorismatase-like hydrolases (IHL) constitute a large family of enzymes divided into five structural families (by SCOP). IHLs are crucial for siderophore-mediated ferric iron acquisition by cells. Knowledge of the structural characteristics of these molecules will enhance the understanding of the molecular basis of iron transport, and perhaps resolve which of the mechanisms previously proposed in the literature is the correct one. We determined the crystal structure of the apo-form of a putative isochorismatase hydrolase OaIHL (PDB code: 3LQY) from the antarctic γ-proteobacterium Oleispira antarctica, and did comparative sequential and structural analysis of its closest homologs. The characteristic features of all analyzed structures were identified and discussed. We also docked isochorismate to the determined crystal structure by in silico methods, to highlight the interactions of the active center with the substrate. The putative isochorismate hydrolase OaIHL from O. antarctica possesses the typical catalytic triad for IHL proteins. Its active center resembles those IHLs with a D-K-C catalytic triad, rather than those variants with a D-K-X triad. OaIHL shares some structural and sequential features with other members of the IHL superfamily. In silico docking results showed that despite small differences in active site composition, isochorismate binds to in the structure of OaIHL in a similar mode to its binding in phenazine biosynthesis protein PhzD (PDB code 1NF8).
Assuntos
Hidrolases/química , Oceanospirillaceae/enzimologia , Cristalografia por Raios X , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
Phosphoglycerate kinase (PGK) is indispensable during glycolysis for anaerobic glucose degradation and energy generation. Here we present comprehensive structure analysis of two putative PGKs from Bacillus anthracis str. Sterne and Campylobacter jejuni in the context of their structural homologs. They are the first PGKs from pathogenic bacteria reported in the Protein Data Bank. The crystal structure of PGK from Bacillus anthracis str. Sterne (BaPGK) has been determined at 1.68 Å while the structure of PGK from Campylobacter jejuni (CjPGK) has been determined at 2.14 Å resolution. The proteins' monomers are composed of two domains, each containing a Rossmann fold, hinged together by a helix which can be used to adjust the relative position between two domains. It is also shown that apo-forms of both BaPGK and CjPGK adopt open conformations as compared to the substrate and ATP bound forms of PGK from other species.
Assuntos
Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Campylobacter jejuni/enzimologia , Fosfoglicerato Quinase/química , Trifosfato de Adenosina/química , Apoenzimas/química , Cristalografia por Raios X , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N(1)-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N(1) atom of a purine residue is discussed.
Assuntos
Adenosina/análogos & derivados , Proteínas Arqueais/metabolismo , Guanosina/análogos & derivados , tRNA Metiltransferases/metabolismo , Adenosina/metabolismo , Proteínas Arqueais/classificação , Biocatálise , Escherichia coli/genética , Guanosina/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Sulfolobus acidocaldarius/enzimologia , Thermococcus/enzimologia , tRNA Metiltransferases/classificaçãoRESUMO
Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 A resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m(7)G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.
Assuntos
Proteínas de Bactérias/química , Metiltransferases/química , RNA Ribossômico 16S/química , Sequência de Aminoácidos , Aminoglicosídeos/farmacologia , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Sequência de Bases , Calorimetria , Domínio Catalítico , Sequência Conservada , Farmacorresistência Bacteriana , Metilação , Micromonospora/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: With the Coronavirus becoming a new reality of our world, global efforts continue to seek answers to many questions regarding the spread, variants, vaccinations, and medications. Particularly, with the emergence of several strains (e.g., Delta, Omicron), vaccines will need further development to offer complete protection against the new variants. It is critical to identify antiviral treatments while the development of vaccines continues. In this regard, the repurposing of already FDA-approved drugs remains a major effort. In this paper, we investigate the hypothesis that a combination of FDA-approved drugs may be considered as a candidate for COVID-19 treatment if (1) there exists an evidence in the COVID-19 biomedical literature that suggests such a combination, and (2) there is match in the clinical trials space that validates this drug combination. METHODS: We present a computational framework that is designed for detecting drug combinations, using the following components (a) a Text-mining module: to extract drug names from the abstract section of the biomedical publications and the intervention/treatment sections of clinical trial records. (b) a network model constructed from the drug names and their associations, (c) a clique similarity algorithm to identify candidate drug treatments. RESULT AND CONCLUSIONS: Our framework has identified treatments in the form of two, three, or four drug combinations (e.g., hydroxychloroquine, doxycycline, and azithromycin). The identifications of the various treatment candidates provided sufficient evidence that supports the trustworthiness of our hypothesis.
RESUMO
Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterised, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains - a small N-terminal beta-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity.
Assuntos
Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Espermidina Sintase/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Calorimetria , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espermidina Sintase/genética , Espermidina Sintase/metabolismo , Especificidade por SubstratoRESUMO
N(1)-methylation of adenosine to m(1)A occurs in several different positions in tRNAs from various organisms. A methyl group at position N(1) prevents Watson-Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m(1)A methylation at position 58 has been identified, while other m(1)A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N(1)-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m(1)A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a DeltatrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m(1)A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m(1)A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m(1)A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , tRNA Metiltransferases/química , Sequência de Aminoácidos , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Bases , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , RNA de Transferência/química , RNA de Transferência/metabolismo , Homologia de Sequência de Aminoácidos , tRNA Metiltransferases/classificação , tRNA Metiltransferases/genéticaRESUMO
Methyltransferases that carry out posttranscriptional N7-methylation of G1405 in 16S rRNA confer bacterial resistance to aminoglycoside antibiotics, including kanamycin and gentamicin. Genes encoding enzymes from this family (hereafter referred to as Arm, for aminoglycoside resistance methyltransferases) have been recently found to spread by horizontal gene transfer between various human pathogens. The knowledge of the Arm protein structure would lay the groundwork for the development of potential resistance inhibitors, which could be used to restore the potential of aminoglycosides to act against the resistant pathogens. We analyzed the sequence-function relationships of Sgm MTase, a member of the Arm family, by limited proteolysis and site-directed and random mutagenesis. We also modeled the structure of Sgm using bioinformatics techniques and used the model to provide a structural context for experimental results. We found that Sgm comprises two domains and we characterized a number of functionally compromised point mutants with substitutions of invariant or conserved residues. Our study provides a low-resolution (residue-level) model of sequence-structure-function relationships in the Arm family of enzymes and reveals the cofactor-binding and substrate-binding sites. These functional regions will be prime targets for further experimental and theoretical studies aimed at defining the reaction mechanism of m7 G1405 methylation, increasing the resolution of the model and developing Arm-specific inhibitors.
Assuntos
Aminoglicosídeos/farmacologia , Farmacorresistência Bacteriana , Metiltransferases/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biologia Computacional , Gentamicinas/farmacologia , Canamicina/farmacologia , Metiltransferases/genética , Micromonospora/classificação , Micromonospora/efeitos dos fármacos , Micromonospora/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
We cloned and sequenced the cspA-like gene from a psychrotrophic Antarctic soil-dwelling bacterial strain Psychrobacter sp. B6. The gene is 213 bp long and shows 99% and 98% sequence identity with the Psychrobacter cryohalolentis K5 gene encoding a cold-shock DNA-binding domain protein and the Psychrobacter arcticus transcriptional regulator-CspA gene, respectively. The protein encoded by the Psychrobacter sp. B6 cspA-like gene shows 100% identity with the two proteins mentioned above, and also 61% sequence identity with CspB from Bacillus subtilis and Csp from Bacillus caldolyticus, and 56% - with Escherichia coli CspA protein. A three-dimensional model of the CspA-like protein from Psychrobacter sp. B6 was generated based on three known structures of cold shock proteins: the crystal structure of the major cold shock protein from Escherichia coli (CspA), the NMR structure of the latter protein, and the NMR structure of Csp from Thermotoga maritima. The deduced structure of the CspA-like protein from Psychrobacter sp. B6 was found to be very similar to these known structures of Csp-like proteins.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Psychrobacter/genética , Microbiologia do Solo , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Conformação Proteica , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 A resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.
Assuntos
Proteínas de Escherichia coli/química , Metiltransferases/química , Modelos Moleculares , Sequência de Aminoácidos , Aminoácidos/química , Biologia Computacional , Proteínas de Escherichia coli/metabolismo , Metiltransferases/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , S-Adenosilmetionina/química , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
In this work, we present the construction of a metagenomic library in Escherichia coli using pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Screening on agar supplemented with olive oil and rhodamine B revealed one clone with lipolytic activity (Lip1) out of 1000 E. coli clones. This clone harbored a plasmid, pLip1, which has an insert of 4722 bp that was completely sequenced from both directions. Further analysis of the insert showed three open reading frames (ORFs). ORF2 encoded a protein (Lip1 ) of 469 amino acids with 93% identity to the uncultured Pseudomonas sp. lipase LipJ03. Amino acid sequence comparison and phylogenetic analysis indicated that Lip1 lipase was closely related to family I subfamily 3. Furthermore, we present a three-dimensional model of lipase Lip1 which was generated based on the two known structures of mesophilic lipases from Pseudomonas sp. MIS 38 (PML lipase, PDB; 2Z8X) and Serratia marcescens (SML lipase, PDB: 2QUB). Finally, we report the results of comparisons between lipase Lip1 and mesophilic lipases and point out similarities and differences in the catalytic site and in other parts of the analyzed structures.
Assuntos
Proteínas de Bactérias/metabolismo , Biblioteca Gênica , Genômica , Lipase/metabolismo , Microbiologia do Solo , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/genética , Lipase/química , Modelos Moleculares , Dados de Sequência MolecularRESUMO
BACKGROUND: SPOUT methyltransferases (MTases) are a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. In 2001, when no crystal structures were available for any of these proteins, Anantharaman, Koonin, and Aravind identified homology between SpoU and TrmD MTases and defined the SPOUT superfamily. Since then, multiple crystal structures of knotted MTases have been solved and numerous new homologous sequences appeared in the databases. However, no comprehensive comparative analysis of these proteins has been carried out to classify them based on structural and evolutionary criteria and to guide functional predictions. RESULTS: We carried out extensive searches of databases of protein structures and sequences to collect all members of previously identified SPOUT MTases, and to identify previously unknown homologs. Based on sequence clustering, characterization of domain architecture, structure predictions and sequence/structure comparisons, we re-defined families within the SPOUT superfamily and predicted putative active sites and biochemical functions for the so far uncharacterized members. We have also delineated the common core of SPOUT MTases and inferred a multiple sequence alignment for the conserved knot region, from which we calculated the phylogenetic tree of the superfamily. We have also studied phylogenetic distribution of different families, and used this information to infer the evolutionary history of the SPOUT superfamily. CONCLUSION: We present the first phylogenetic tree of the SPOUT superfamily since it was defined, together with a new scheme for its classification, and discussion about conservation of sequence and structure in different families, and their functional implications. We identified four protein families as new members of the SPOUT superfamily. Three of these families are functionally uncharacterized (COG1772, COG1901, and COG4080), and one (COG1756 represented by Nep1p) has been already implicated in RNA metabolism, but its biochemical function has been unknown. Based on the inference of orthologous and paralogous relationships between all SPOUT families we propose that the Last Universal Common Ancestor (LUCA) of all extant organisms contained at least three SPOUT members, ancestors of contemporary RNA MTases that carry out m1G, m3U, and 2'O-ribose methylation, respectively. In this work we also speculate on the origin of the knot and propose possible 'unknotted' ancestors. The results of our analysis provide a comprehensive 'roadmap' for experimental characterization of SPOUT MTases and interpretation of functional studies in the light of sequence-structure relationships.
Assuntos
Biologia Computacional , Evolução Molecular , Metiltransferases/química , Metiltransferases/genética , Família Multigênica , Sequência de Aminoácidos , Animais , Humanos , Metiltransferases/classificação , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA) biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM) domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM). However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. RESULTS: Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase) fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. CONCLUSION: The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily), but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.
Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , MicroRNAs/biossíntese , Filogenia , Plantas/enzimologia , Plantas/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Evolução Molecular , Metiltransferases/genética , Dados de Sequência Molecular , Família Multigênica , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
BACKGROUND: Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT. RESULTS: As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases. CONCLUSION: Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).
Assuntos
Proteínas de Escherichia coli/fisiologia , tRNA Metiltransferases/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células Eucarióticas , Metilação , Células Procarióticas , RNA de Transferência/metabolismo , Terminologia como Assunto , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismoRESUMO
In the course of CASP6, we generated models for all targets using a new version of the "FRankenstein's monster approach." Previously (in CASP5) we were able to build many very accurate full-atom models by selection and recombination of well-folded fragments obtained from crude fold recognition (FR) results, followed by optimization of the sequence-structure fit and assessment of alternative alignments on the structural level. This procedure was however very arduous, as most of the steps required extensive visual and manual input from the human modeler. Now, we have automated the most tedious steps, such as superposition of alternative models, extraction of best-scoring fragments, and construction of a hybrid "monster" structure, as well as generation of alternative alignments in the regions that remain poorly scored in the refined hybrid model. We have also included the ROSETTA method to construct those parts of the target for which no reasonable structures were generated by FR methods (such as long insertions and terminal extensions). The analysis of successes and failures of the current version of the FRankenstein approach in modeling of CASP6 targets reveals that the considerably streamlined and automated method performs almost as well as the initial, mostly manual version, which suggests that it may be a useful tool for accurate protein structure prediction even in the hands of nonexperts.
Assuntos
Biologia Computacional/métodos , Proteômica/métodos , Algoritmos , Automação , Simulação por Computador , Computadores , Bases de Dados de Proteínas , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Alinhamento de Sequência , SoftwareRESUMO
We present the crystal structures of two universal stress proteins (USP) from Archaeoglobus fulgidus and Nitrosomonas europaea in both apo- and ligand-bound forms. This work is the first complete synthesis of the structural properties of 26 USP available in the Protein Data Bank, over 75% of which were determined by structure genomics centers with no additional information provided. The results of bioinformatic analyses of all available USP structures and their sequence homologs revealed that these two new USP structures share overall structural similarity with structures of USPs previously determined. Clustering and cladogram analyses, however, show how they diverge from other members of the USP superfamily and show greater similarity to USPs from organisms inhabiting extreme environments. We compared them with other archaeal and bacterial USPs and discuss their similarities and differences in context of structure, sequential motifs, and potential function. We also attempted to group all analyzed USPs into families, so that assignment of the potential function to those with no experimental data available would be possible by extrapolation.
RESUMO
2'-O-ribose methylation is one of the most common posttranscriptional modifications in RNA. Methylations at different positions are introduced by enzymes from at least two unrelated superfamilies. Recently, a new family of eukaryotic RNA methyltransferases (MTases) has been identified, and its representative from yeast (Yol125w, renamed as Trm13p) has been shown to 2'-O-methylate position 4 of tRNA. Trm13 is conserved in Eukaryota, but exhibits no sequence similarity to other known MTases. Here, I present the results of bioinformatics analysis which suggest that Trm13 is a strongly diverged member of the Rossmann-fold MTase (RFM) superfamily, and therefore is evolutionarily related to 2'-O-MTases such as Trm7 and fibrillarin. However, the character of conserved residues in the predicted active site of the Trm13 family suggests it may use a different mechanism of ribose methylation than its relatives. A molecular model of the Trm13p structure has been constructed and evaluated for potential accuracy using model quality assessment methods. The predicted structure will facilitate experimental analyses of the Trm13p mechanism of action.