ENZYME-MEDIATED CYTOSINE DEAMINATION BY THE BACTERIAL METHYLTRANSFERASE M.MSPI

Most prokaryotic (cytosine-5)-DNA methyltransferases increase the freq uency of deamination at the cytosine targeted for methylation in vitro in the absence of the cofactor S-adenosylmethionine (AdoMet) or the r eaction product S-adenosylhomocysteine (AdoHcy). We show here that, un der the same in vitro conditions, the prokaryotic methyltransferase, M .MspI (from Moraxella sp.), causes very few cytosine deaminations, sug gesting a mechanism in which M.MspI may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet, sinefungin and 5'-amino-5'-deox yadenosine, greatly increased the frequency of cytosine deamination me diated by M.MspI presumably by introducing a proton-donating amino gro up into the catalytic centre, thus facilitating the formation of an un stable enzyme-dihydrocytosine intermediate and hydrolytic deamination. Interestingly, two naturally occurring analogues, adenosine and 5'-me thylthio-5'-deoxyadenosine, which do not contain a proton-donating ami no group, also weakly increased the deamination frequency by M.MspI, e ven 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 hydrol ytic deamination of the enzyme-dihydrocytosine intermediate. Under nor mal physiological conditions the enzymes M.HpaII (from Haemophilus par ainfluenzae), M.HhaI (from Haemophilus hemolytica) and M.MspI all incr eased the in vivo deamination frequency at the target cytosines with c omparable efficiency.