Discovery of a bacterial 5-methylcytosine deaminase.
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 × 10(5), 2.9 × 10(4), and 1.1 × 10(3) 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 × 10(5), 6.8 × 10(4), and 2.0 × 10(2) 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.
Department of Biochemistry & Biophysics and ‡Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
Daniel S
DS
Hitchcock
Alexander A
AA
Fedorov
Elena V
EV
Fedorov
Steven C
SC
Almo
Frank M
FM
Raushel
D044503:5-Methylcytosine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D055162:Biocatalysis; D020134:Catalytic Domain; D002460:Cell Line; D043525:Cytosine Deaminase / Q000737:chemistry / Q000378:metabolism*; D005437:Flucytosine / Q000378:metabolism / Q000633:toxicity; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D013941:Thymine / Q000378:metabolism
D044503:5-Methylcytosine; D005437:Flucytosine; D043525:Cytosine Deaminase; D013941:Thymine