Functional Analysis of Conserved Motifs inEcoP15I DNA Methyltransferase

Ahmad, Ishtiyaque; Rao, Desirazu N.

doi:10.1006/jmbi.1996.0315

Cited by 33 publications

(27 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These variants are mutated at the following amino acid residues that are impli- Our results suggest that all of these contacts to the cofactor observed in the x-ray structure analyses of several methyltransferases (M. HhaI (7), catechol-O-methyltransferase (43), M. TaqI (28), HaeIII DNA methyltransferase (15), VP39 vaccinia protein RNA methyltransferase (44), CheR (45), and PvuII DNA methyltransferase (29)) are very important for binding and positioning the cofactor. The data obtained with Glu 37 and Phe 39 in motif I (FXGXG) complement other mutational studies concerned with this region, because exchanging the first glycine of the FXGXG by other amino acid residues prevents AdoMet binding by EcoKI and EcoP15I (23,24).…”

Section: Implications Of the Results On The Structural Model For A-typesupporting

confidence: 78%

“…It should be noted that variants that are not able to flip out the target base are not expected to have a lower affinity toward DNA, because nonspecific binding of M. EcoRV to DNA is almost as strong as specific binding (34 (25). In contrast, a DPPY 3 DPPW exchange in EcoP15I inactivates this enzyme (24).…”

Section: Implications Of the Results On The Structural Model For A-typementioning

confidence: 99%

“…Consequently, these additional motifs only recently could be identified in structure-guided alignments (21). Whereas some amino acid residues within the FXGXG and DPPY motifs have already been investigated by site-directed mutagenesis (22)(23)(24)(25), no mutational studies have been carried out so far to test the functional roles of conserved amino acid residues within the other motifs either in C-or N-MTases.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Roth

Helm-Kruse

Friedrich

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Section: Implications Of the Results On The Structural Model For A-typesupporting

confidence: 78%

Section: Implications Of the Results On The Structural Model For A-typementioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Roth

Helm-Kruse

Friedrich

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…Substituting tyrosine in motif IV of EcoP15I DNA MTase by site-directed mutagenesis resulted in loss of enzyme activity although we observed enhanced cross-linking of AdoMet and DNA. These results reinforce the importance of motif IV in catalysis (20).…”

supporting

confidence: 81%

“…We have recently demonstrated that altering amino acid residues in the motif I of EcoP15I DNA MTase resulted in loss of AdoMet binding but left DNA target recognition unaltered (20). A second motif characteristic of m6A-MTases and m4C-MTases, (N/ D/S) PP (Y/F) (motif IV) (21), is well conserved in EcoP15I DNA MTase.…”

mentioning

confidence: 99%

Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

Reddy

Rao

1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5,5-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.EcoP15I DNA methyltransferase (EcoP15I DNA MTase) 1 catalyzes the transfer of a methyl group from S-adenosyl-Lmethionine (AdoMet) to the second adenine nucleotide in the canonical site 5Ј-CAGCAG-3Ј (1) to form N 6 -methyladenine. The enzyme is part of the type III restriction-modification (R-M) system (2). Type III R-M enzymes are multifunctional proteins that exert both methylation and restriction activities (2). Type III R-M systems contain two subunits, the Res subunit encoded by the res gene and the Mod subunit encoded by the mod gene. Although the Mod subunit alone can catalyze the methylation reaction, both the Res and Mod subunits are necessary for DNA cleavage (2). The enzymes have an absolute requirement for ATP for restriction, and recently we and others (3, 4) showed that ATP hydrolysis was required for DNA cleavage. It has been shown that only the Mod subunit is involved in DNA sequence recognition in both the restriction and modification reactions (5). We had earlier shown by gel mobility shift assays that EcoP15I DNA MTase binds about 3-fold more tightly to DNA containing its recognition sequence 5Ј-CAG-CAG-3Ј than to nonspecific sequences in the absence or presence of cofactors. Interestingly, in the presence ...

show abstract