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, it was 
shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI and M.MspI 
is enhanced in the presence of several analogues of SAM, and a mechanism for 
this analogue-promoted deamination was proposed. According to this 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 protonation of C5. Further, we show 
that uracil is an intermediate in the mutational pathway and propose an 
alternate mechanism for the analogue-promoted deamination. 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 mechanism was tested by using M.EcoRII 
mutants defective in the nucleophilic attack at C6 in the deamination assay. We 
find that although these proteins are defective in methyl transfer and cytosine 
deamination, they cause cytosine deaminations in the presence of AA in the 
reaction. Our results point to a possible connection between the catalytic 
mechanism of C5 Mtases and of enzymes that transfer methyl groups to N(4) of 
cytosine. Further, they provide an unusual example where a coenzyme activates an 
otherwise "dead" enzyme to perform catalysis by a new reaction pathway.