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

20 Citations (Scopus)

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

Fingerprint

Cytosine Deaminase
Deamination
Catalyst activity
Cytosine
Zinc
Substrates
Chemical activation
Amidohydrolases
Reaction intermediates
Mutation
Kinetics
Proton transfer
Uracil
Water
Divalent Cations
Enzymes
Isotopes
Protons
Hydrogen
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

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

Three-dimensional structure and catalytic mechanism of cytosine deaminase. / Hall, Richard S.; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 50, No. 22, 07.06.2011, p. 5077-5085.

Research output: Contribution to journalArticle

Hall, RS, Fedorov, AA, Xu, C, Fedorov, EV, Almo, SC & Raushel, FM 2011, 'Three-dimensional structure and catalytic mechanism of cytosine deaminase', Biochemistry, vol. 50, no. 22, pp. 5077-5085. https://doi.org/10.1021/bi200483k
Hall, Richard S. ; Fedorov, Alexander A. ; Xu, Chengfu ; Fedorov, Elena V. ; Almo, Steven C. ; Raushel, Frank M. / Three-dimensional structure and catalytic mechanism of cytosine deaminase. In: Biochemistry. 2011 ; Vol. 50, No. 22. pp. 5077-5085.
@article{34eee96be8b2436f8887343bc0df43a7,
title = "Three-dimensional structure and catalytic mechanism of cytosine deaminase",
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.",
author = "Hall, {Richard S.} and Fedorov, {Alexander A.} and Chengfu Xu and Fedorov, {Elena V.} and Almo, {Steven C.} and Raushel, {Frank M.}",
year = "2011",
month = "6",
day = "7",
doi = "10.1021/bi200483k",
language = "English (US)",
volume = "50",
pages = "5077--5085",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "22",

}

TY - JOUR

T1 - Three-dimensional structure and catalytic mechanism of cytosine deaminase

AU - Hall, Richard S.

AU - Fedorov, Alexander A.

AU - Xu, Chengfu

AU - Fedorov, Elena V.

AU - Almo, Steven C.

AU - Raushel, Frank M.

PY - 2011/6/7

Y1 - 2011/6/7

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=79958058125&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79958058125&partnerID=8YFLogxK

U2 - 10.1021/bi200483k

DO - 10.1021/bi200483k

M3 - Article

C2 - 21545144

AN - SCOPUS:79958058125

VL - 50

SP - 5077

EP - 5085

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 22

ER -