Discovery of a bacterial 5-methylcytosine deaminase

Daniel S. Hitchcock, Alexander A. Fedorov, Elena V. Fedorov, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 105, 2.9 × 104, and 1.1 × 103 M-1 s-1, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 105, 6.8 × 104, and 2.0 × 102 M-1 s-1, respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85), 5-fluorocytosine (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.

Original languageEnglish (US)
Pages (from-to)7426-7435
Number of pages10
JournalBiochemistry
Volume53
Issue number47
DOIs
StatePublished - Dec 2 2014

ASJC Scopus subject areas

  • Biochemistry

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  • Cite this

    Hitchcock, D. S., Fedorov, A. A., Fedorov, E. V., Almo, S. C., & Raushel, F. M. (2014). Discovery of a bacterial 5-methylcytosine deaminase. Biochemistry, 53(47), 7426-7435. https://doi.org/10.1021/bi5012767