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Journal of Virology, August 2005, p. 9945-9953, Vol. 79, No. 15
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.15.9945-9953.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Chlorella Virus-Encoded Deoxyuridine Triphosphatases Exhibit Different Temperature Optima

Department of Plant Pathology, University of Nebraska-Lincoln, Nebraska 68583-0722,¹ Department of Chemistry, University of Nebraska-Lincoln, Nebraska 68583-0304,² Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68588-0666,³ Nebraska Center of Virology, University of Nebraska, Lincoln, Nebraska 68588-0666⁴

Received 26 January 2005/ Accepted 15 April 2005

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 K_m of 11.7 µM, a turnover k_cat of 6.8 s^–1, and a catalytic efficiency of k_cat/K_m = 5.8 x 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, Glu81Ser81 and Thr84Arg84, 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: Thr84Arg84, Glu81Ser81, and Glu81Ser81 plus Thr84Arg84. 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.

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