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Structural and Biochemical Studies on the Reaction Mechanism of Uridine–Cytidine Kinase

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Abstract

Uridine–cytidine kinase catalyzes phosphorylation of the pyrimidine nucleosides uridine and cytidine and plays an important role in nucleotide metabolism. However, the detailed molecular mechanism of these reactions remains to be elucidated. Here, we determined the structure of the ternary complex of Uridine–cytidine kinase from Thermus thermophilus HB8 with both cytidine and β,γ-methyleneadenosine 5′-triphosphate, a non-hydrolysable ATP analogue. Substrate binding is accompanied by substantial domain movement that allows the substrate-binding cleft to close. The terminal phosphodiester bond of the ATP analogue is in an ideal location for an inline attack of the 5′-hydroxyl group of cytidine. Asp40 is located near the 5′-hydroxyl group of cytidine. Mutation of this conserved residue to Asn or Ala resulted in a complete loss of enzyme activity, which is consistent with the notion that Asp40 acts as a general base that activates the 5′-hydroxyl group of cytidine. The pH profile of the activity showed an apparent pK a value of 7.4. Based on this structure, a likely mechanism of the catalytic step is discussed.

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Abbreviations

AMPPCP:

β,γ-methyleneadenosine 5′-triphosphate

CMP:

Cytidine monophosphate

cv:

Column volume

NMP:

Nucleoside monophosphate

ttUCK:

Uridine–cytidine kinase in T. thermophilus HB8

UCK:

Uridine–cytidine kinase

WT:

Wild type

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Acknowledgments

This work was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Challenging Exploratory Research 25650008 (to RM) and the Japan Society for the Promotion of Science for Young Scientists (10J0180400-00, FT).

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Author notes

  1. Fumiaki Tomoike

    Present address: Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan

Authors and Affiliations

  1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Fumiaki Tomoike & Seiki Kuramitsu

  2. Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan

    Noriko Nakagawa, Seiki Kuramitsu & Ryoji Masui

  3. Division of Biology and Geosciences, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka, 558-8585, Japan

    Ryoji Masui

Authors
  1. Fumiaki Tomoike
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  2. Noriko Nakagawa
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  3. Seiki Kuramitsu
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  4. Ryoji Masui
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Corresponding author

Correspondence to Ryoji Masui.

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Tomoike, F., Nakagawa, N., Kuramitsu, S. et al. Structural and Biochemical Studies on the Reaction Mechanism of Uridine–Cytidine Kinase. Protein J 34, 411–420 (2015). https://doi.org/10.1007/s10930-015-9636-8

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