N6-methyladenine and
N6,
N6-dimethyladenine are the heterocyclic bases present in the RNA of eukaryotic and bacterial cells and play important regulatory roles. How the degradation of such modified nucleic acids, and the subsequent demethylation of modified heterocyclic bases, occurs in the bacterium
Escherichia coli is not established. Here, we investigated the growth of adenine auxotroph strains in a minimal M9 medium supplemented with either
N6-methyladenine or
N6,
N6-dimethyladenine. We found that
N6-methyladenine supported the growth of
∆purH::Km but not that of the
∆purA::Km strain, whereas
N6,
N6-dimethyladenine did not support the growth of either adenine auxotroph. Similar experiments performed using structurally related 2-amino-
N6-methylpurine and 2-amino-
N6,
N6-dimethylpurine bases—using
∆guaA::Km,
∆guaB::Km, and
∆purH::Km guanine auxotrophs—demonstrated that growth of only the
∆guaB::Km mutant was supported by 2-amino-
N6-methylpurine but not by its dimethylated counterpart. We expressed and purified C-teminus 6xHis tagged
E. coli adenine/adenosine deaminases AdeC and Add and tested their substrate specificity. We demonstrated that AdeC protein does not catalyse deamination of either
N6-methyl- or
N6,
N6-dimethyladenine, whereas Add catalyses deamination of
N6-methyl- but not that of
N6,
N6-dimethyladenosine. Based on our findings, biochemical pathways leading to the demodification and return into metabolism of
N6-methyladenine and 2-amino-
N6-methylpurine in
E. coli are proposed.
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