title: A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine.
abstract: The salvage pathways of nucleotide biosynthesis are more diverse and are less well understood as compared with de novo pathways. Uridine-cytidine kinase (UCK) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. In this study, we have characterized a UCK homologue of Thermus thermophilus HB8 (ttCK) biochemically and structurally. Unlike other UCKs, ttCK had substrate specificity toward only cytidine and showed no inhibition by UTP, suggesting uridine does not bind to ttCK as substrate. Structural analysis revealed that the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK. Replacement of Tyr93 by histidine or glutamine endowed ttCK with phosphorylation activity toward uridine. These results suggested that a single amino acid residue, Tyr93, gives cytidine-limited specificity to ttCK. However, replacement of Tyr93 by Phe or Leu did not change the substrate specificity of ttCK. Therefore, we conclude that a residue at this position is essential for the recognition of uridine by UCK. In addition, thymidine phosphorylase from T. thermophilus HB8 was equally active with thymidine and uridine, which indicates that this protein is the sole enzyme metabolizing uridine in T. Thermophilus HB8. On the basis of these results, we discuss the pyrimidine-salvage pathway in T. thermophilus HB8.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Tomoike
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakagawa
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Ryoji
authors.initials: R
authors.lastname: Masui
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003562:Cytidine / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*; D014541:Uridine Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D003562:Cytidine; D014541:Uridine Kinase