Reviving a dead enzyme: Cytosine deaminations promoted by an inactive DNA methyltransferase and an S-adenosylmethionine analogue

The enzymes that transfer a methyl group to C5 of cytosine within specific sequences (C5 Mtases) deaminate the target cytosine to uracil if the methyl donor S-adenosylmethionine (SAM) is omitted from the reaction. Recently, i t was shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI a nd M.MspI is enhanced in the presence of several analogues of SAM, and a me chanism for this analogue-promoted deamination was proposed. According to t his mechanism, the analogues protonate C5 of the target cytosine, creating a dihydrocytosine intermediate that is susceptible to deamination. We show here that one of these analogues, 5'-aminoadenosine (AA), enhances cytosine deamination by the Mtase M.EcoRII, but it does so without enhancing proton ation of C5. Further, we show that uracil is an intermediate in the mutatio nal pathway and propose an alternate mechanism for the analogue-promoted de amination. The new mechanism involves a facilitated water attack at C4 but does not require attack at C6 by the enzyme. The latter feature of the mech anism was tested by using M.EcoRII: mutants defective in the nucleophilic a ttack at C6 in the deamination assay. We find that although these proteins are defective in methyl transfer and cytosine deamination, they cause cytos ine deaminations in the presence of AA in the reaction. Our results point t o a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer methyl groups to N-4 Of cytosine. Further, they prov ide an unusual example where a coenzyme activates an otherwise "dead" enzym e to perform catalysis by a new reaction pathway.