Structural and Biochemical Studies of Thermus Thermophilus HB8 Uridine-Cytidine Kinase on Urd/Cyt analogs

Abstract

Pyrimidine nucleotide salvage pathway is conserved in most living organisms, where a pyrimidine nucleoside is phosphorylated at the 5’-hydroxyl group to generate a corresponding monophosphate form. Uridine/cytidine kinase (UCK) usually displays a dual specificity towards both uridine and cytidine, which converts uridine or cytidine to UMP or CMP, respectively. Meanwhile, an ortholog from Thermus thermophilus HB8 (ttCK) is able to phosphorylate cytidine only, but not uridine. This limited specificity arises from the single amino acid residue substitution of otherwise highly conserved histidine with tyrosine at position 93. Although this unique functional property has been well demonstrated by kinetic analyses, structural basis underlying how His-93 substitution allows the mutant ttCK to phosphorylate uridine remains undetermined. Additionally, whether genetically engineered ttCK can act on a bulky pyrimidine nucleoside has not been extensively exploited. In order to address these issues, we have attempted to create mutant ttCKs intended to act on uridine/cytidine derivatives, including 5-methoxyuridine (mo5U), and 5-methylcytidine (m5C). Among those, crystal structures of Y43A, Y93H, and Y43A/Y93H were determined to 1.95, 2.60, and 2.30 Å, respectively, and the catalytic activity of the mutant enzymes against various pyrimidine nucleosides were examined. Together with steady-state kinetics of mutant enzymes, structure-function relationship of ttCK could be derived at atomic level.