Three-dimensional structure and catalytic mechanism of cytosine deaminase

Richard S. Hall, Alexander A. Fedorov, Chengfu Xu, Elena V. Fedorov, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a Ki of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on kcat and kcat/Km, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

Original languageEnglish (US)
Pages (from-to)5077-5085
Number of pages9
JournalBiochemistry
Volume50
Issue number22
DOIs
StatePublished - Jun 7 2011

ASJC Scopus subject areas

  • Biochemistry

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    Hall, R. S., Fedorov, A. A., Xu, C., Fedorov, E. V., Almo, S. C., & Raushel, F. M. (2011). Three-dimensional structure and catalytic mechanism of cytosine deaminase. Biochemistry, 50(22), 5077-5085. https://doi.org/10.1021/bi200483k