Functional Analysis of Conserved Motifs in Type III. Restriction-Modification Enzymes

Abstract

EcoP1I and EcoP15I are members of type III restriction-modification enzymes. EcoPI and EcoP15I DNA methyltransferases transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the second adenine residues in their recognition sequences, 5'-AGACC-3' and 5'-CAGCAG-3' respectively. We have altered various residues in two highly conserved sequences, FxGxG (motif I) and DPPY (motif IV) in these proteins by site-directed mutagenesis. Using a mixture of in vivo and in vitro assays, our results on the mutational analysis of these methyltransferases demonstrate the universal role of motif I in AdoMet binding and a role for motif IV in catalysis. All six cysteine residues in EcoP15I DNA methyltransferase have been substituted with serine and the role of cysteine residues in EcoP15I DNA methyltransferase catalysed reaction assessed. The Res subunits of type III restriction enzymes share a distant sequence similarity with and contain the motifs characteristic of the DEAD box proteins. We have carried out site-directed mutagenesis of the conserved residues in two of the helicase motifs of the EcoP1I restriction enzyme in order to investigate the role of motifs in DNA cleavage by this enzyme. Our findings indicate that certain conserved residues in these motifs are involved in ATP hydrolysis while the other residues are involved in coupling restriction of DNA to ATP hydrolysis. Taken collectively, these results form the basis for a detailed structure-function analysis of EcoP1I and EcoP15I restriction enzymes.

Transcriptional Phase Variation of a Type III Restriction-Modification System in Helicobacter pylori

“…No clear homologs are present in the genome of H. pylori strain 26695 (37). The conserved motifs involved in AdoMet binding, catalysis, and ATP-dependent reactions, which are characteristic for all type III RM systems (31), are present in the putative translated products of the mod and the res genes. It is therefore likely that the RM system identified in this study is a type III RM system.…”

“…DNA cleavage requires a complex of both the subunits (19) and occurs at a specific site approximately 25 bp to one side of the recognition site (24). Type III methyltransferases typically contain two conserved motifs involved in AdoMet binding and catalysis (31). The presence of conserved DEAD box helicase motifs, involved in the ATP-dependent reactions of the enzyme, is characteristic of type III restriction enzymes (31).…”

“…Type III methyltransferases typically contain two conserved motifs involved in AdoMet binding and catalysis (31). The presence of conserved DEAD box helicase motifs, involved in the ATP-dependent reactions of the enzyme, is characteristic of type III restriction enzymes (31).…”

Molecular Characterization of an rsmD -Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme

“…Two motifs, viz. the SAM binding motif (FXGXG) as well as the catalytic motif (DPPY), are present in the enzyme sequence for proper catalysis of the methylation reaction (24)(25)(26), with the RNA recognition sequence being located in the stretch of 8 to 26 amino acids at the N terminus, as predicted (54). The latter information conforms to the fact that the naked RNA is copurified along with the enzyme, which gets methylated on addition of SAM-3H (data not shown); however, there is still a possibility that it has methylation activity on the assembled 30S subunit from an rsmD mutant strain, and this possibility should be examined.…”

“…Due to its high level of importance in ribosome translation events, this enzyme has now been considered a novel drug target (11,23). Here we report on the molecular characterization of an rsmD-like methyltransferase from the Wolbachia endosymbiont of B. malayi (the wBmrRNA MTase) and the use of specific inhibitors against its catalytic motif, DPPY (24)(25)(26). Methyltransferases with the DPPY motif are not present in higher eukaryotes; rather, most of their methyltransferases have the CCWGG motif for C-5 cytosine methylation (27)(28)(29)(30).…”