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1.  New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA 
Nucleic Acids Research  2010;38(19):6533-6543.
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 N1-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 N1 atom of a purine residue is discussed.
PMCID: PMC2965216  PMID: 20525789
2.  Assessing the Accuracy of Template-Based Structure Prediction Metaservers by Comparison with Structural Genomics Structures 
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 ( and 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.
PMCID: PMC4105012  PMID: 23086054
3.  Structural and functional insight into the universal stress protein family 
Evolutionary Applications  2013;6(3):434-449.
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.
PMCID: PMC3673472  PMID: 23745136
Archaeoglobus fulgidus; crystal structures; Nitrosomonas europaea; pathogens; sequence analyses; structural comparison; structural genomics; universal stress protein
4.  Crystal structure of a putative isochorismatase hydrolase from Oleispira antarctica 
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 solved crystal structure by in silico methods, to highlight the interactions of the active center with the substrate.
The putative isochorismate hydrolase OaIHL from Oleispira 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).
PMCID: PMC3328404  PMID: 22350524
iron uptake; isochorismatase hydrolases; structural comparison of isochorismatases; structural genomics
5.  Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases 
Nucleic Acids Research  2010;38(12):4120-4132.
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 Å 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 m7G 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.
PMCID: PMC2896518  PMID: 20194115
6.  The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK) 
Nucleic Acids Research  2008;36(10):3252-3262.
N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 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 m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-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(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. 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 m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.
PMCID: PMC2425500  PMID: 18420655
7.  Functional specialization of domains tandemly duplicated within 16S rRNA methyltransferase RsmC 
Nucleic Acids Research  2007;35(13):4264-4274.
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 Å 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.
PMCID: PMC1934991  PMID: 17576679
8.  Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases 
BMC Bioinformatics  2007;8:73.
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.
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.
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.
PMCID: PMC1829167  PMID: 17338813
9.  The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase 
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.
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.
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).
PMCID: PMC1569432  PMID: 16848900
10.  Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis 
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.
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.
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.
PMCID: PMC1397878  PMID: 16433904

Results 1-10 (10)