title: Molecular mechanisms of substrate specificities of uridinecytidine kinase.
abstract: A uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine (Urd) and cytidine (Cyd) and plays a significant role in the pyrimidine-nucleotide salvage pathway. Unlike ordinary ones, UCK fromThermus thermophilusHB8 (ttCK) loses catalytic activity on Urd due to lack of a substrate binding ability and possesses an unusual amino acid, i.e. tyrosine 93 (Tyr93) at the binding site, whereas histidine (His) is located in the other UCKs. Mutagenesis experiments revealed that a replacement of Tyr93 by His or glutamine (Gln) recovered catalytic activity on Urd. However, the detailed molecular mechanism of the substrate specificity has remained unclear. In the present study, we performed molecular dynamics simulations on the wild-type ttCK, two mutant ttCKs, and a human UCK bound to Cyd and three protonation forms of Urd to elucidate their substrate specificity. We found three residues, Tyr88, Tyr/His/Gln93 and Arg152 in ttCKs, are important for recognizing the substrates. Arg152 contributes to induce a closed form of the binding site to retain the substrate, and the N3 atom of Urd needed to be deprotonated. Although Tyr88 tightly bound Cyd, it did not sufficiently bind Urd because of lack of the hydrogen bonding. His/Gln93 complemented the interaction of Tyr88 and raised the affinity of ttCK to Urd. The crucial distinction between Tyr and His or Gln was a role in the hydrogen-bonding network. Therefore, the ability to form both hydrogen-bonding donor and accepter is required to bind both Urd and Cyd.
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Tanaka
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan; Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Shoji
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Tomoike
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan.
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Ujiie
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan.
authors.forename: Kyohei
authors.initials: K
authors.lastname: Hanaoka
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan.
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Harada
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan; Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Kayanuma
authors.affiliation: Center for Basic Education and Integrated Learning, Kanagawa Institute of Technology, Atsugi, Kanagawa 243-0292, Japan.
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Kamiya
authors.affiliation: Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Toyokazu
authors.initials: T
authors.lastname: Ishida
authors.affiliation: Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Masui
authors.affiliation: Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.affiliation: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan; Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Shigeta
keywords: binding free energy; deprotonated; hydrogen bond; keto-enol-enolate; molecular dynamics