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J M Bujnicki

Publications and source records attributed to J M Bujnicki.

35 records · Page 2Linked to original sources
PubMed2001

Cloning of enterohemorrhagic Escherichia coli phage VT-2 dam methyltransferase.

Enterobacterial GATC-specific DNA adenine methyltransferase (Dam) plays an essential role in regulation of DNA replication, methyl-directed mismatch repair, transposition and gene expression. In Salmonella typhimurium it has been shown to directly control virulence. In this paper we report cloning and expression of the dam gene from the Shiga toxin-producing VT2-Sa prophage of enterohemorrhagic Escherichia coli O157. Comparisons of the predicted amino acid sequence indicates that Dam methyltransferases of E. coli phages VT2-Sa, 933W, T1 and Haemophilus influenzae phage HP1 make up a separate subgroup of adenine-N6 methyltransferases. These proteins are similar to the gamma subfamily of amino-methyltransferases in respect to the linear order of sequence motifs and the presence of the hallmark "NPPY" tetrapeptide. However, they apparently lack an autonomous target-recognizing domain at the C-terminus of the catalytic domain and therefore we propose to dub them as a "mini-gamma" subfamily.

Amino Acid Motifs↗
PubMed2001

Site-directed mutagenesis defines the catalytic aspartate in the active site of the atypical DNA: m4C methyltransferase M.NgoMXV.

M.NgoMXV is one of the few atypical DNA:m4C methyltransferases that does not possess a serine residue in its predicted active site. We previously reported a homology model of M.NgoMXV and argued that the aspartate side chain at a corresponding position, similarly to some DNA:m6 A-specific enzymes, is essential for the methyltransferase activity (Radlinska et al., 1999). Here we report the corrected amino acid sequence of M.NgoMXV and the analysis of substitution of D68 with alanine or serine, which both render the enzyme totally inactive.

Amino Acid Sequence↗
PubMed2001

A model of structure and action of Sau3AI restriction endonuclease that comprises two MutH-like endonuclease domains within a single polypeptide.

Sau3AI is a type II endonuclease that cleaves GATC sequences, producing sticky ends with 4-nucleotide 5'-overhangs. Its activity is inhibited by cytosine C5-methylation within the target sequence. In the N-terminus, Sau3AI exhibits sequence similarity to the GATC-specific single-strand nicking endonuclease MutH implicated in mismatch repair (Ban and Yang, 1998). Sequence analysis of Sau3AI and its homologs reveals that Sau3AI possesses an additional MutH-like domain in the C-terminus. Structure prediction suggests that the C-terminal domain lacks the endonuclease active site but retains all putative DNA-binding elements. As an illustration of these findings, a model of quaternary structure of Sau3AI complexed with the target DNA is presented. These predictions have implications for evolution, structure and function of bacterial DNA repair enzymes and restriction endonucleases.

Amino Acid Sequence↗
PubMed2001

Understanding the evolution of restriction-modification systems: clues from sequence and structure comparisons.

Restriction-modification (RM) systems comprise two opposing enzymatic activities: a restriction endonuclease, that targets specific DNA sequences and performs endonucleolytic cleavage, and a modification methyltransferase that renders these sequences resistant to cleavage. Studies on molecular genetics and biochemistry of RM systems have been carried out over the past four decades, laying foundations for modern molecular biology and providing important models for mechanisms of highly specific protein-DNA interactions. Although the number of known, relevant sequences 3D structures of RM proteins is growing steadily, we do not fully understand their functional diversities from an evolutionary perspective and we are not yet able to engineer new sequence specificities based on rational approaches. Recent findings on the evolution of RM systems and on their structures and mechanisms of action have led to a picture in which conserved modules with defined function are shared between different RM proteins and other enzymes involved in nucleic acid biochemistry. On the other hand, it has been realized that some of the modules have been replaced in the evolution by unrelated domains exerting similar function. The aim of this review is to give a survey on the recent progress in the field of structural phylogeny of RM enzymes with special emphasis on studies of sequence-structure-function relationships and emerging potential applications in biotechnology.

Catalytic Domain↗
PubMed2001

Cloning of the Haemophilus influenzae Dam methyltransferase and analysis of its relationship to the Dam methyltransferase encoded by the HP1 phage.

In this paper we report cloning and experimental characterization of the DNA adenine methyltransferase (dam) gene from Haemophilus influenzae and comparison of its product with the Dam protein from the lysogenic phage of H. influenzae, HP1. Molecular modeling of M.HinDam and M.HP1Dam was carried out, providing a framework for a comparative analysis of these enzymes and their close homologs in the structural context. Both proteins share the common fold and essential cofactor-binding and catalytic residues despite overall divergence. However, subtle but significant differences in the cofactor-binding pocket have been identified. Moreover, while M.HinDam seems to contact its target DNA sequence using a number of loops, most of them are missing from M.HP1Dam. Analysis of both MTases suggests that their catalytic activity was derived from a common ancestor, but similar sequence specificities arose by convergence.

Amino Acid Motifs↗
PubMed2000

Homology modelling of the DNA 5mC methyltransferase M.BssHII. Is permutation of functional subdomains common to all subfamilies of DNA methyltransferases?

This work presents a full tertiary model of the M.BssHII methyltransferase (MTase) complexed with substrate DNA and cofactor S-adenosyl-L-methionine, built by homology modelling based on previously solved complete structures of DNA MTases M.HaeIII and M. HhaI. M.BssHII and the template proteins show high sequence similarity, which indicates that they are evolutionary related. However, they are topologically different: M.BssHII is a circularly permuted variant of template MTases (Xu et al. Nucleic Acids Res 1997;25:3991). The model developed in this work will be a good starting point and valuable help in designing mutagenesis experiments to better understand the biological function of methyltransferases and the process of domain swapping.

Amino Acid Sequence↗
PubMed2000

Phylogeny of the restriction endonuclease-like superfamily inferred from comparison of protein structures.

To date all attempts to derive a phyletic relationship among restriction endonucleases (ENases) from multiple sequence alignments have been limited by extreme divergence of these enzymes. Based on the approach of Johnson et al. (1990), I report for the first time the evolutionary tree of the ENase-like protein superfamily inferred from quantitative comparison of atomic coordinates of structurally characterized enzymes. The results presented are in harmony with previous comparisons obtained by crystallographic analyses. It is shown that lambda-exonuclease initially diverged from the common ancestor and then two "endonucleolytic" families branched out, separating "blunt end cutters" from "5' four-base overhand cutters." These data may contribute to a better understanding of ENases and encourage the use of structure-based methods for inference of phylogenetic relationship among extremely divergent proteins. In addition, the comparison of three-dimensional structures of ENase-like domains provides a platform for further clustering analyses of sequence similarities among different branches of this large protein family, rational choice of homology modeling templates, and targets for protein engineering.

Catalytic Domain↗
PubMed2000

Phylogenomic analysis of 16S rRNA:(guanine-N2) methyltransferases suggests new family members and reveals highly conserved motifs and a domain structure similar to other nucleic acid amino-methyltransferases.

The sequences of known Escherichia coli 16S rRNA:m2G1207 methyltransferase (MTase) RsmC and hypothetical 16S rRNA:m2G966 MTase encoded by the ygjo open reading frame were used to carry out a database search of other putative m2G-generating enzymes in finished and unfinished genomic sequences. Sequence comparison and phylogenetic analysis of 21 close homologs of RsmC and YgjO revealed the presence of the third paralogous lineage in E. coli and other gamma-Proteobacteria, which might correspond to the subfamily of MTases specific for G1516 in 16S rRNA. In addition, the comparative sequence analysis supported by sequence/structure threading suggests that rRNA:m2G MTases are very closely related to RNA and DNA:m6A MTases and that these two enzyme families share common architecture of the active site and presumably a similar mechanism of methyl group transfer onto the exocyclic amino group of their target bases.

Amino Acid Sequence↗
PubMed2000

Sequence, structural, and evolutionary analysis of prokaryotic ribosomal protein L11 methyltransferases.

The Escherichia coli PrmA enzyme catalyzes methylation of the large ribosomal subunit protein L11. Database homology searches, multiple sequence alignment, and structure prediction allowed to dissect the primary structure of PrmA into two domains and assign putative functional or structural roles to invariant or highly conserved residues. Evolutionary relationships within the PrmA family were also analyzed. The topology of the branching order agrees to a large extent with the consensus phylogeny of Eubacteria, with the exception of beta and epsilon subdivisions of Proteobacteria, which most probably had their original prmA genes replaced by copies acquired via the lateral gene transfer from gamma-Proteobacteria and some close relative of the ancestor of gramnegative bacteria, respectively.

Amino Acid Sequence↗
PubMed2000

Prediction of a novel RNA 2'-O-ribose methyltransferase subfamily encoded by the Escherichia coli YgdE open reading frame and its orthologs.

The amino acid sequence of the RNA 2'-O-ribose methyltranserase RrmJ was used as a probe for detecting putative homologs through iterative searches of genomic databases. We found a previously unannotated YgdE open reading frame (ORF) in the genome sequences of Escherichia coli and other gamma-Proteobacteria, which shares key features with RrmJ, despite the mutual sequence similarity of these proteins is relatively low. The predicted structural compatibility and the conservation of all functionally important residues between RrmJ and YgdE strongly suggests that the newly identified methyltranserase also modifies 2'-OH groups of ribose. The N-terminal region of YgdE, which has no counterpart in RrmJ, is predicted to form an independent domain, possibly involved in target recognition.

Amino Acid Sequence↗
PubMed1999

Structural characterization of two tandemly arranged DNA methyltransferase genes from Neisseria gonorrhoeae MS11: N4-cytosine specific M.NgoMXV and nonfunctional 5-cytosine-type M.NgoMorf2P.

Two adjacent genes encoding DNA methyltransferases (MTases) of Neisseria gonorrhoeae MS11, an active N4-cytosine specific M. NgoMXV and an inactive 5-cytosine type M. NgoMorf2P, were cloned into Escherichia coli and sequenced. We analyzed the deduced amino acid sequence of both gene products and localized conserved regions characteristic for DNA MTases. Structure prediction, threading-derived alignments, and comparison with the common fold for DNA MTases allowed for construction of super-secondary and tertiary models for M.NgoMorf2P and M.NgoMXV, respectively. These models helped in identification of amino acids and structural elements essential for function of both enzymes. The implications of this putative structural model on the catalytic mechanism of M.NgoMXV and its possible relation to the common ancestor of modern DNA amino-MTases are also discussed.

Amino Acid Sequence↗
PubMed1999

Molecular evolution of DNA-(cytosine-N4) methyltransferases: evidence for their polyphyletic origin.

DNA N4-cytosine methyltransferases (N4mC MTases) are a family of S-adenosyl-L-methionine (AdoMet)-dependent MTases. Members of this family were previously found to share nine conserved sequence motifs, but the evolutionary basis of these similarities has never been studied in detail. We performed phylogenetic analysis of 37 known and potential new family members from the multiple sequence alignment using distance matrix, parsimony and maximum likelihood approaches to infer the evolutionary relationship among the N4mC MTases and classify them into groups of orthologs. All the treeing algorithms employed as well as results of exhaustive sequence database searching support a scenario, in which the majority of N4mC MTases, except for M. Bal I and M. Bam HI, arose by divergence from a common ancestor. Interestingly, MTases M. Bal I and M. Bam HI apparently originated from N6-adenine MTases and represent the most recent addendum to the N4mC MTase family. In addition to the previously reported nine sequence motifs, two more conserved sequence patches were detected. Phylogenetic analysis also provided the evidence for massive horizontal transfer of MTase genes, presumably with the whole restriction-modification systems, between Bacteria and Archaea.

Amino Acid Sequence↗
PubMed1999

Is the HemK family of putative S-adenosylmethionine-dependent methyltransferases a "missing" zeta subfamily of adenine methyltransferases? A hypothesis.

Previous comparative studies revealed close similarity among various groups of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases), indicating their common evolutionary origin. We present evidence for a remarkable similarity between the sequence and predicted structure of HemK (a widespread family of putative proteins encoded in genomes from bacteria to humans) and the catalytic domain of the gamma-subfamily of adenine-specific DNA MTases (N6mA MTases). We predict the structure and function of the putative catalytic domain of HemK proteins and speculate that the target-recognizing function may be conferred by the N-terminal variable region.

Amino Acid Sequence↗
PubMed1999

Molecular phylogenetics of DNA 5mC-methyltransferases.

We have identified a total of 88 members of the DNA-(cytosine-5) methyltransferase (5mC MTase) family whose sequences have been deposited in the databases. The results of a comparison of these sequences is presented in the form of an alignment-based phylogenetic tree and sequence logos for 10 conserved motifs. Phylogenetic analysis showed that members of the family aggregate into subfamilies which are usually consistent with their target specificity. However, it was also shown that similar target specificity does not necessarily imply close homology of the catalytic domain of MTases, which strongly supports the hypothesis that target recognition evolved independently of catalytic properties. This analysis also indicate that the 5mC MTase was present in the cenancestor (last common ancestor) of eubacteria, archaebacteria, and eukaryotes. The phylogeny of the 5mC MTases catalytic domain provides the basis for establishing the patterns of evolutionary change that characterize this family of proteins with conserved structural core and variable and mobile modules not directly involved in formation of the active site.

Amino Acid Sequence↗
PubMedDate not supplied

Comparison of protein structures reveals monophyletic origin of the AdoMet-dependent methyltransferase family and mechanistic convergence rather than recent differentiation of N4-cytosine and N6-adenine DNA methylation.

Phylogenetic analysis of the S-adenosyl-L-methionine-dependent methyltransferases was performed based on similarity of positions of main chain alpha-carbon atoms in published structures of members of this superfamily. The evolutionary tree was inferred and the problem of mono/polyphyletic origin of DNA methyltransferases from the Rossmann-fold enzymes was solved, bridging two seemingly antithetical hypotheses. The comparison of protein structures provides evidence for an evolutionary link between widely diverged subfamilies of RNA and DNA N6-adenine methyltransferases and argues against the close homology of N6-adenine and N4-cytosine methyltransferases, apparent from biochemical data and comparison of fragments of sequences. Such evolutionary analysis of methyltransferases has never been published yet in the literature and will guide further phylogenetical studies based on both sequence and structure comparison.

Adenine↗