Mutagenesis and biochemical characterization of signature motifs in Type III RM enzyme

Abstract

Restriction-modification (RM) system in bacteria has evolved to protect bacterial cell from foreign DNA that enters the cell. Type III RM system is one of the classes of RM system involved in such a defense mechanism. It is comprised of a Mod and a Res subunit that has methylation and restriction function respectively. Though the Type III system is extensively studied through EcoP15I and EcoP1I enzymes, many structural and mechanistic details are still to be known. In this study, mutations and biochemical characterization have been carried out in hope to understand the important motifs for methylation activity. A highly conserved stretch GDN in EcoP15I was characterized by mutation and found to be involved in the binding of the cofactor for methylation. Structural analysis of different classes of methyltransferases has shown that a presence of a negative charge in the cofactor binding pocket is a characteristic feature of all methyltransferases. We propose that this particular GDN motif in EcoP15I is the actual Motif III in Type III RM enzyme, which is assigned wrongly at present and is important in the SAM (S-adenosyl methionine, the cofactor for methylation) binding.

Characterization of the MboIII protein was done to find the functional relevance of a loop in the methyltransferase domain. In contrast to the hypothesis proposed, the residues in the loop were found to be important in the restriction activity and not methylation activity. Based on the data obtained, we suggest that the loop T128 to S156 is an allosteric regulator for the function of Res subunit. The function of this loop could be replaced by the binding of the ligand in the cofactor pocket of methyltransferase. Together with the bound ligand and an ordered loop that interacts to the ligand, arrays of interactions are initiated from the Mod subunit all the way up to the Res subunit resulting in the cleavage of DNA. Detailed experiments have to be carried out to characterize this long-range interaction.