Rescue of the orphan enzyme isoguanine deaminase

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

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: kcat = 49 s-1, Km = 72 μM, and kcat/Km = 6.7 × 105 M-1 s -1. The kinetic constants for the deamination of cytosine are as follows: kcat = 45 s-1, Km = 302 μM, and kcat/Km = 1.5 × 105 M-1 s -1. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

Original languageEnglish (US)
Pages (from-to)5555-5557
Number of pages3
JournalBiochemistry
Volume50
Issue number25
DOIs
StatePublished - Jun 28 2011

ASJC Scopus subject areas

  • Biochemistry

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    Hitchcock, D. S., Fedorov, A. A., Fedorov, E. V., Dangott, L. J., Almo, S. C., & Raushel, F. M. (2011). Rescue of the orphan enzyme isoguanine deaminase. Biochemistry, 50(25), 5555-5557. https://doi.org/10.1021/bi200680y