Enzyme-mediated cytosine deamination by the bacterial methyltransferase M.MspI.
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.
Zingg
J M
JM
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles, CA 90033, USA.
Shen
J C
JC
Jones
P A
PA
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis; D003641:Deamination; D003839:Deoxyadenosines / Q000378:metabolism; D015394:Molecular Structure; D009016:Moraxella / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D009355:Neomycin / Q000737:chemistry; D012435:S-Adenosylhomocysteine / Q000031:analogs & derivatives / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D003839:Deoxyadenosines; C057928:5'-amino-5'-deoxyadenosine; D012436:S-Adenosylmethionine; D003596:Cytosine; D012435:S-Adenosylhomocysteine; D004248:DNA (Cytosine-5-)-Methyltransferases; D009355:Neomycin; D000241:Adenosine; C006235:sinefungin