This is the first reported structure of a prokaryotic UCK. The overall structure is highly similar to that of human uridine cytidine kinase 2 (UCK2). Structural comparison of the nucleoside-binding site between ttCK and human UCK2 revealed that most amino acid residues around the base moieties are identical between them, except for Tyr59 and Tyr93 in ttCK, corresponding to Phe83 and His117 in human UCK2. These two residues are located near the N4 amino group and the N3 nitrogen atom of cytosine: these atoms differ between cytosine and uracil. Many UCK homologues have a Phe and His residue at the position equivalent to Tyr59 and Tyr93 in ttCK, respectively. As the next step, we prepared ttCK mutants with amino acid substitution at Tyr59 and Tyr93. The substrate specificity of Y59F was the same as that for wild type. In contrast, Y93H had activity for both uridine and cytidine. Whereas the replacement of Tyr93 with Phe or Leu, unaffected the substrate specificity of ttCK, the replacement with Gln, Asn and Glu endowed ttCK with phosphorylation activity toward uridine. These results indicate that a hydrophilic residue at position 93 permits UCK to accept uridine as substrate. A potential hydrogen donor at this position may enable UCK to interact a keto group of uridine specifically. In addition to ttCK, other UCK homologues of 26 species including pathogens have tyrosine at the position equivalent to Tyr93, which predicts that these are cytidine-specific UCKs. This study points to the critical need for experimental studies even of enzymes whose annotation has been accepted.
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