Abstract
Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.
Highlights
Deoxycytidylate deaminases are the only allosterically regulated members of the zinc-dependent cytidine deaminase family
Deoxycytidylate Deaminase Isolated from S-TIM5 Functions as a Typical dCMP Deaminase—S-TIM5 is a recently isolated cyanophage that infects Synechococcus and was characterized as uniquely belonging to the Myoviridae family [19]
Recombinant S-TIM5-dCD was found to function in a manner typical to dCMP deaminases that are strictly controlled by dCTP activation: in solution, it exists in the hexameric form and catalyzes deoxycytidine deamination only on the dCMP substrate and only in the presence of dCTP allosteric activator (Fig. 1B, lines a and b)
Summary
Deoxycytidylate deaminases are the only allosterically regulated members of the zinc-dependent cytidine deaminase family. Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. We present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is discussed. The atomic coordinates and structure factors (codes 4P9E, 4P9D, and 4P9C) have been deposited in the Protein Data Bank (http://wwpdb.org/)
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