摘要
5-Hydroxymethylcytosine (5hmC) was present in T-even phage and mammalian DNA. 5hmC in phage is formed by hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) by deoxycytidylate hydroxymethylase (CH), which uses the solvent water as the hydroxyl group donor. By contrast, 5hmC is formed in mammal zygotes by the oxidation of 5-methylcytosine (5mC). 5hmC was also present in a nucleoside antibiotic mildiomycin and its formation is governed by a cytidylate hydroxymethylase MilA. However, the catalytic mechanism remains unknown. In the present study, we purified His-tagged MilA and fed its in vitro reaction with H218O. The LC-MS analysis of the product revealed that 18O was incorporated into the hydroxymethylated CMP (HmCMP), and the secondary MS result of 18O-labeled HmCMP indicated that 18O was incorporated into the cytosine of HmCMP. The results demonstrate that MilA uses solvent water as the hydroxyl group donor like CH. Moreover, Thr102 of MilA was predicted as potential critical amino acid anchoring one molecule of water for hydroxylation. Finally, organizational context comparison in microbial genomes reveals that six homologous ORFs originally annotated as putative thymidylate synthase (TS) are more likely to be CMP hydroxymethylase.
5-Hydroxymethylcytosine （5hmC） was present in T-even phage and mammalian DNA. 5hmC in phage is formed by hydroxymethylation of the cytosine base in deoxycytidylate （dCMP） by deoxycytidylate hydroxymethylase （CH）, which uses the solvent water as the hydroxyl group donor. By contrast, 5hmC is formed in mammal zygotes by the oxidation of 5-methylcytosine （5mC）. 5hmC was also present in a nucleoside antibiotic mildiomycin and its formation is governed by a cytidylate hydroxymethylase MilA. However, the catalytic mechanism remains unknown. In the present study, we purified His-tagged MilA and fed its in vitro reaction with H2^18O. The LC-MS analysis of the product revealed that ^18O was incorporated into the hydroxymethylated CMP （HmCMP）, and the secondary MS result of ^18O-labeled HmCMP indicated that ^18O was incorporated into the cytosine of HmCMP. The results demonstrate that MilA uses solvent water as the hydroxyl group donor like CH. Moreover, Thr102 of MilA was predicted as potential critical amino acid anchoring one molecule of water for hydroxylation. Finally, organizational context comparison in microbial genomes reveals that six homologous ORFs originally annotated as putative thymidylate synthase （TS） are more likely to be CMP hydroxymethylase.
基金
supported by the National Natural Science Foundation of China (31170083)
the Ministry of Science and Technology (2012CB721004)
the Shanghai Municipal Council of Science and Technology
