Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima.
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 Mg(2+) 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 microM, a turnover k(cat) of 6.8 s(-1), and a catalytic efficiency of k(cat)/K(m) = 5.8 x 10(5) 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 degrees C) than PBCV-1 dUTPase (50 degrees 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 degrees 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.
Zhang
Yuanzheng
Y
Department of Plant Pathology, University of Nebraska-Lincoln, 68583-0722, USA.
Moriyama
Hideaki
H
Homma
Kohei
K
Van Etten
James L
JL
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002708:Chlorella / Q000821:virology*; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017862:Phycodnaviridae / Q000201:enzymology / Q000235:genetics*; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D011994:Recombinant Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase