title: Reviving a dead enzyme: cytosine deaminations promoted by an inactive DNA methyltransferase and an S-adenosylmethionine analogue.
abstract: 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.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
authors.forename: A N
authors.initials: AN
authors.lastname: Sharath
authors.forename: E
authors.initials: E
authors.lastname: Weinhold
authors.forename: A S
authors.initials: AS
authors.lastname: Bhagwat
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000409:Alanine / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D003596:Cytosine / Q000737:chemistry*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics; D003641:Deamination; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D012436:S-Adenosylmethionine / Q000737:chemistry*; D012694:Serine / Q000235:genetics; D014498:Uracil / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D012694:Serine; D014498:Uracil; D012436:S-Adenosylmethionine; D003596:Cytosine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases; D000241:Adenosine; D003545:Cysteine; D000409:Alanine