Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

EcoRI DNA methyltransferase contains tryptophans at positions 183 and 225. Tryptophan 225 is adjacent to residues previously implicated in S-adenosylmethionine (AdoMet) binding and to cysteine 223, previously shown to be the site of N-ethyl maleimide-mediated inactivation of the enzyme (Reich, N. O., and Everett, E. (1990) J. Biol. Chem. 265, 8929-8934; Everett, E. A., Falick, A. M., and Reich, N. O. (1990) J. Biol. Chem. 265, 17713-17719). The fluorescence spectra of the wild-type enzyme is centered at 338 nm indicating partial tryptophan solvent accessibility. Substitution of tryptophan 183 with phenylalanine results in a 45% drop in fluorescence intensity, but no shift in lambda max. DNA binding to the wild-type methyltransferase caused an increase in the fluorescence intensity, while binding to the tryptophan 183 mutant had a quenching effect, suggesting that DNA binding induces a conformational change near both tryptophans. Binding of AdoMet and various AdoMet analogs to the wild-type methyltransferase results in no change in the fluorescence spectrum when excitation occurs at 295 nm, suggesting that no conformational change occurs, and AdoMet does not interact with either tryptophan. In contrast, quenching was observed when excitation occurred at 280 nm, suggesting that AdoMet and its analogs may be quenching tyrosine to tryptophan energy transfer. Protein-ligand complexes were titrated with acrylamide, and the data also implicate conformational changes upon DNA binding but not upon AdoMet binding, consistent with previous limited proteolysis results (Reich, N. O., Maegley, K. A., Shoemaker, D.D., and Everett, E. (1991) Biochemistry 30, 2940-2946).
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PMID:Cofactor and DNA interactions in EcoRI DNA methyltransferase. Fluorescence spectroscopy and phenylalanine replacement for tryptophan 183. 152 89

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.
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PMID:Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents. 174 39

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.
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PMID:Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. 861 77

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue.
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PMID:Functional analysis of conserved motifs in EcoP15I DNA methyltransferase. 865 25

The adenine-specific DNA methyltransferase M.TaqI transfers a methyl group from S-adenosylmethionine to N6 of the adenine residue in the DNA sequence 5'-TCGA-3'. In the crystal structure of M.TaqI in complex with S-adenosylmethionine the enzyme is folded into two domains: An N-terminal catalytic domain, whose fold is conserved among S-adenosyl-methionine dependent methyltransferases, and a DNA recognition domain which possesses a unique fold. In the active site, two aromatic residues, Tyr 108 and Phe 196, are postulated to bind the flipped-out target DNA adenine which becomes methylated. By lowering the energy of the positively charged transition state via cationic-pi interactions, these two residues probably hold a key role in catalysis.
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PMID:M.TaqI: possible catalysis via cation-pi interactions in N-specific DNA methyltransferases. 962 29

Cytosine (C-5)-specific DNA methyltransferases share a set of ten conserved motifs distributed evenly throughout the entire polypeptide chain. The first conserved motif contains a Phe, which is intimately associated with cofactor recognition. In the pseudo-DNA methyltransferase M.SpoI, encoded by the pmt1 gene in Schizosaccharomyces pombe, a Tyr replaces this Phe residue. We describe the properties of a mutant form of M.MspI, a typical cytosine (C-5)-specific DNA methyltransferase, in which Tyr replaces the conserved Phe. This mutant shows differences in ternary complex formation and in the pattern of covalent complex formation with an inhibitory, fluorinated DNA duplex which may be due to anomalous hydrogen bonding between the mutant Tyr hydroxyl group and the catalytic loop of the enzyme or through interference with cofactor binding.
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PMID:Substitution of the conserved phenylalanine in the S-adenosyl-L-methionine binding site of M.MspI with tyrosine modifies the kinetic properties of the enzyme. 962 62

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.
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PMID:Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus. 993 Oct 7

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.
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PMID:Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry. 1111 26

Protein tyrosine phosphatases (PTPs) are comprised of two superfamilies, the phosphatase I superfamily containing a single low-molecular-weight PTP (lmwPTP) family and the phosphatase II superfamily including both the higher-molecular-weight PTP (hmwPTP) and the dual-specificity phosphatase (DSP) families. The phosphatase I and II superfamilies are often considered to be the result of convergent evolution. The PTP sequence and structure analyses indicate that lmwPTPs, hmwPTPs, and DSPs share similar structures, functions, and a common signature motif, although they have low sequence identities and a different order of active sites in sequence or a circular permutation. The results of this work suggest that lmwPTPs and hmwPTPs/DSPs are remotely related in evolution. The earliest ancestral gene of PTPs could be from a short fragment containing about 90 approximately 120 nucleotides or 30 approximately 40 residues; however, a probable full PTP ancestral gene contained one transcript unit with two lmwPTP genes. All three PTP families may have resulted from a common ancestral gene by a series of duplications, fusions, and circular permutations. The circular permutation in PTPs is caused by a reading frame difference, which is similar to that in DNA methyltransferases. Nevertheless, the evolutionary mechanism of circular permutation in PTP genes seems to be more complicated than that in DNA methyltransferase genes. Both mechanisms in PTPs and DNA methyltransferases can be used to explain how some protein families and superfamilies came to be formed by circular permutations during molecular evolution.
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PMID:Different protein tyrosine phosphatase superfamilies resulting from different gene reading frames. 1267 37


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