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.

Mononuclear Iron Enzymes Are Primary Targets of Hydrogen Peroxide Stress

“…PDF purifies with bound zinc and was originally believed to be a zinc enzyme, but the instability of the just-extracted enzyme in aerobic buffers matches the instability of the iron-constituted enzyme (23). CDA purified from cells is predominantly occupied by zinc with a minor amount of iron, and mechanistic studies have usually employed the zinc-loaded enzyme; however, kinetic efficiency is somewhat higher with iron (24,25). TDH exhibits good activity when constituted with zinc and is tentatively regarded as using zinc as an active-site metal in vivo; it is less active with manganese, and activity with iron has not been reported (26,27).…”

Nucleobase deaminases: a potential enzyme system for new therapies

“…In the active site, H246 and D313 are poised to serve as general acid/base groups to activate the metal-bound water molecule and the amino leaving group. 17,22 In addition, E217 is positioned to deliver the proton to the N3 atom of the pyrimidine ring. In addition, the carbamoyl moiety at N1/C2 is stabilized and hydrogen bonded with the side chain of Q156 (Fig.…”

“…5 Nucleobase deamination is catalysed via metal-assisted hydrolysis of amino groups of heterocyclic bases in nucleosides, nucleotides and nucleic acids. 12,17 Nature has evolved a variety of deaminases; based on their structural folds, these enzymes are categorized into two major sub-groups: the amidohydrolase (AHS) and cytidine deaminase-like (CDA) superfamilies. 9,12 Although both these families use metal-assisted deamination as their primary mode of catalysis, they show extreme sequence and structural divergence.…”

Characterization and Cytotoxic Activity of Cytosine Deaminase Enzyme Purified from Locally Isolated Escherichia coli

“…Cytosine deaminase (CD, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively (5). The enzyme is gave a wide interest for antimicrobial drug design and for gene therapy application against cancer.…”