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 Citations (Scopus)

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 k<inf>cat</inf>/K<inf>m</inf> of 1.4 × 10<sup>5</sup>, 2.9 × 10<sup>4</sup>, and 1.1 × 10<sup>3</sup> M<sup>-1</sup> s<sup>-1</sup>, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of k<inf>cat</inf>/K<inf>m</inf> of 1.2 × 10<sup>5</sup>, 6.8 × 10<sup>4</sup>, and 2.0 × 10<sup>2</sup> M<sup>-1</sup> s<sup>-1</sup>, 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

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5-Methylcytosine
Thymine
Flucytosine
Escherichia coli
Cytosine Deaminase
Enzymes
Fluorouracil
Catalytic Domain
Corynebacterium glutamicum
Rhodobacter sphaeroides
Deamination
Poisons
Klebsiella pneumoniae
Sequence Homology
Eukaryota
X-Ray Diffraction
X ray diffraction
Substrates
Growth

ASJC Scopus subject areas

  • Biochemistry

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

Discovery of a bacterial 5-methylcytosine deaminase. / Hitchcock, Daniel S.; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 53, No. 47, 02.12.2014, p. 7426-7435.

Research output: Contribution to journalArticle

Hitchcock, DS, Fedorov, AA, Fedorov, EV, Almo, SC & Raushel, FM 2014, 'Discovery of a bacterial 5-methylcytosine deaminase', Biochemistry, vol. 53, no. 47, pp. 7426-7435. https://doi.org/10.1021/bi5012767
Hitchcock DS, Fedorov AA, Fedorov EV, Almo SC, Raushel FM. Discovery of a bacterial 5-methylcytosine deaminase. Biochemistry. 2014 Dec 2;53(47):7426-7435. https://doi.org/10.1021/bi5012767
Hitchcock, Daniel S. ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Almo, Steven C. ; Raushel, Frank M. / Discovery of a bacterial 5-methylcytosine deaminase. In: Biochemistry. 2014 ; Vol. 53, No. 47. pp. 7426-7435.
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