Functional Roles of Conserved Amino Acid Residues in DNA Methyltransferases Investigated by Site-directed Mutagenesis of theEcoRV Adenine-N6-methyltransferase

Roth, Markus; Helm-Kruse, Sabine; Friedrich, Tatjana; Jeltsch, Albert

doi:10.1074/jbc.273.28.17333

Cited by 64 publications

(73 citation statements)

References 45 publications

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“…1, B and C); this is nearly universal to class I MTases (13). These interactions have been experimentally shown to be essential for AdoMet binding for many DNA MTases, including the bacterial M.EcoRV (24) and M.HhaI enzymes (25) and the murine Dnmt3a enzymes (22). A unique interaction of EcoDam involves the indole group of Trp 10 , whose ring nitrogen forms a hydrogen bond with one of the ribose hydroxyls (Fig.…”

Section: Resultsmentioning

confidence: 99%

Two Alternative Conformations of S-Adenosyl-L-homocysteine Bound to Escherichia coli DNA Adenine Methyltransferase and the Implication of Conformational Changes in Regulating the Catalytic Cycle

Liebert

Horton

Chahar

et al. 2007

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

Two Alternative Conformations of S-Adenosyl-L-homocysteine Bound to Escherichia coli DNA Adenine Methyltransferase and the Implication of Conformational Changes in Regulating the Catalytic Cycle

Liebert

Horton

Chahar

et al. 2007

Journal of Biological Chemistry

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“…45,46 Interestingly, the main catalytic residue in m 6 which is proposed to stabilize the cationic transition state by cation-π interaction. 49,50 And the second one is the aromatic residue Y/F/W of motif VIII, which stabilizes the flipped base outside the DNA helix. 49,50 These aromatic residues are conserved in motifs IV and VIII in m 1 A tRNA MTases (Figure 7(a), symbol o), but they are not part of the catalytic pocket in the TrmI structure.…”

Section: Superimposition Of T Thermophilus and M Tuberculosismentioning

confidence: 99%

“…49,50 And the second one is the aromatic residue Y/F/W of motif VIII, which stabilizes the flipped base outside the DNA helix. 49,50 These aromatic residues are conserved in motifs IV and VIII in m 1 A tRNA MTases (Figure 7(a), symbol o), but they are not part of the catalytic pocket in the TrmI structure. Strikingly, two other aromatic residues, i.e.…”

Section: Superimposition Of T Thermophilus and M Tuberculosismentioning

confidence: 99%

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Barraud

Golinelli‐Pimpaneau

Atmanène

et al. 2008

Journal of Molecular Biology

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“…All MTases possess a similar catalytic domain and D/NPPY/F sequence, which represents one of nine described motifs (I to VIII and X) and is the common conserved catalytic motif in region IV of adenine-N 6 and cytosine-C (29). To verify that alteration of amino acid residues within or outside the conserved catalytic DPPY active domain abolishes the MTase activity of A. hydrophila, we examined E. coli GM33 strains containing pBAD/dam plasmids with different amino acid mutations within the dam gene.…”

Section: Resultsmentioning

confidence: 99%

“…The intent of the present study was to show whether the decreased virulence of A. hydrophila following Dam overproduction could be attributed to a direct increase in the MTase activity associated with Dam. To address this question, we mutated aspartic acid (D) and tyrosine (Y) residues individually within the Dam catalytic DPPY motif (region IV) by in vitro site-directed mutagenesis based on earlier studies with various MTases (9,29).…”

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confidence: 99%

Mutations within the Catalytic Motif of DNA Adenine Methyltransferase (Dam) of Aeromonas hydrophila Cause the Virulence of the Dam-Overproducing Strain To Revert to That of the Wild-Type Phenotype

et al. 2006

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In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/dam D/A , pBAD/dam Y/A , and pBAD/dam AhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (damdeficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/dam AhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD 50 s) of the WT bacterium. Importantly, immunization of mice with a native-Dam-overproducing strain at the same LD 50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.Aeromonads are ubiquitous organisms which exist in aquatic environments, various types of foods, such as meat, fish and vegetables, and the intestines of apparently healthy humans with diarrhea (11). Among the various Aeromonas species, Aeromonas hydrophila is most commonly involved in human infections, such as septicemia and gastroenteritis (3, 7). The pathogenesis of A. hydrophila infection is complex and multifactorial and characterized by the involvement of a number of virulence factors (1).Our laboratory recently extensively characterized a type 2 secretion system (T2SS)-secreted cytotoxic enterotoxin, Act, and two cytotonic enterotoxins, Alt (heat labile) and Ast (heat stable), from a diarrheal isolate, SSU, of A. hydrophila (31). We provided evidence that ferric uptake regulator (Fur) and glucose-inhibited division A protein (GidA) regulated Act levels at the transcriptional and translational levels, respectively (32, 33). All of these three enterotoxins contributed significantly to the causation of fluid...

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Functional Roles of Conserved Amino Acid Residues in DNA Methyltransferases Investigated by Site-directed Mutagenesis of theEcoRV Adenine-N6-methyltransferase

Cited by 64 publications

References 45 publications

Two Alternative Conformations of S-Adenosyl-L-homocysteine Bound to Escherichia coli DNA Adenine Methyltransferase and the Implication of Conformational Changes in Regulating the Catalytic Cycle

Two Alternative Conformations of S-Adenosyl-L-homocysteine Bound to Escherichia coli DNA Adenine Methyltransferase and the Implication of Conformational Changes in Regulating the Catalytic Cycle

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Mutations within the Catalytic Motif of DNA Adenine Methyltransferase (Dam) of Aeromonas hydrophila Cause the Virulence of the Dam-Overproducing Strain To Revert to That of the Wild-Type Phenotype

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