Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima

Yuanzheng Zhang, Hideaki Moriyama, Kohei Homma, James L. Van Etten

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg2+ for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a Km of 11.7 μM, a turnover kcat of 6.8 s-1, and a catalytic efficiency of kcat/Km = 5.8 × 105 M -1 s-1. dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37°C) than PBCV-1 dUTPase (50°C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81→Ser81 and Thr84→Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84→Arg84, Glu81→Ser81, and Glu81→Ser81 plus Thr84→Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55°C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.

Original languageEnglish (US)
Pages (from-to)9945-9953
Number of pages9
JournalJournal of virology
Volume79
Issue number15
DOIs
StatePublished - Aug 2005

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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