Cytosine methyltransferase from Escherichia coli in which active site cysteine is replaced with serine is partially active.
EcoRII methyltransferase (M.EcoRII) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine (SAM) to C-5 position of second cytosine in the DNA sequence 5'-CCWGG (W = A or T). The reaction is initiated by a nucleophilic attack of the C-6 of target cytosine by a cysteine that is conserved among all cytosine methyltransferases. We have replaced this cysteine in M.EcoRII with serine or alanine and purified the proteins to homogeneity. The catalytic efficiency (kcat/Km) of the mutant enzyme with serine (C186S) for methyl transfer is about 10,000 times less than that of WT but is substantially higher than the efficiency of the C186A mutant. We show that the WT enzyme and C186S mutant are proficient in exchange of proton at C-5 and that this activity is reduced in the mutant to the same extent as the methyl transfer activity. The C186S mutant is insensitive to a cysteine-specific inhibitor, and it transfers methyl groups to the same position of cytosine as the WT enzyme. The ability of serine to act as a nucleophile in the enzyme reaction suggests that it--and probably the cysteine in the WT enzyme--is activated by a nearby base. Like the WT enzyme, C186S forms stable SDS-resistant complexes with DNA containing 5-azacytosine; but unlike the WT enzyme, the mutant reacts faster with 5-azacytosine than with normal cytosine. Apparently, greater reactivity of 5-azacytosine assists the C186S mutant in catalysis.
Gabbara
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S
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Sheluho
D
D
Bhagwat
A S
AS
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000235:genetics*; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D012694:Serine / Q000235:genetics*
D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; C039854:5-azacytosine; D012694:Serine; D003596:Cytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; D003545:Cysteine