Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of 
deamination at the cytosine targeted for methylation in vitro in the absence of 
the cofactor S-adenosylmethionine (AdoMet) or the reaction product 
S-adenosylhomocysteine (AdoHcy). We show here that, under the same in vitro 
conditions, the prokaryotic methyltransferase, M.MspI (from Moraxella sp.), 
causes very few cytosine deaminations, suggesting a mechanism in which M.MspI 
may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet, 
sinefungin and 5'-amino-5'-deoxyadenosine, greatly increased the frequency of 
cytosine deamination mediated by M.MspI presumably by introducing a 
proton-donating amino group into the catalytic centre, thus facilitating the 
formation of an unstable enzyme-dihydrocytosine intermediate and hydrolytic 
deamination. Interestingly, two naturally occurring analogues, adenosine and 
5'-methylthio-5'-deoxyadenosine, which do not contain a proton-donating amino 
group, also weakly increased the deamination frequency by M.MspI, even in the 
presence of AdoMet or AdoHcy. These analogues may trigger a conformational 
change in the enzyme without completely inhibiting the access of solvent water 
to the catalytic centre, thus allowing hydrolytic deamination of the 
enzyme-dihydrocytosine intermediate. Under normal physiological conditions the 
enzymes M.HpaII (from Haemophilus parainfluenzae), M. HhaI (from Haemophilus 
hemolytica) and M.MspI all increased the in vivo deamination frequency at the 
target cytosines with comparable efficiency.