Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Deoxyuridine triphosphatase (dUTPase) is crucial for DNA synthesis and repair by preventing the incorporation of uracil into DNA.
• Chlorella viruses encode dUTPases, but their biochemical properties and structure-function relationships are not fully understood.

Purpose of the Study:

• To clone, express, and characterize dUTPase enzymes from chlorella viruses PBCV-1, IL-3A, and SH-6A.
• To investigate the molecular basis for differences in temperature optima between PBCV-1 and IL-3A dUTPases using computational and mutagenesis approaches.

Main Methods:

• Recombinant protein expression in Escherichia coli.
• Enzyme activity assays, including kinetic studies (K(m), k(cat)) and determination of optimal conditions (Mg(2+), temperature).
• Homology modeling, docking simulations, and site-directed mutagenesis.

Main Results:

• Recombinant PBCV-1 dUTPase exhibited optimal activity with Mg(2+), with kinetic parameters K(m) = 11.7 microM and k(cat) = 6.8 s(-1).
• IL-3A dUTPase showed a lower temperature optimum (37°C) compared to PBCV-1 dUTPase (50°C), attributed to specific amino acid differences in conserved motif III.
• Site-directed mutagenesis, particularly the double substitution Glu81-->Ser81 and Thr84-->Arg84 in IL-3A dUTPase, significantly increased the temperature optimum to 55°C.

Conclusions:

• Chlorella virus dUTPases are biochemically active enzymes requiring divalent cations for optimal function.
• Specific amino acid substitutions, particularly in conserved regions, can significantly alter enzyme thermal stability and kinetics.
• Changes in active site charge and bulkiness, influenced by amino acid composition, are key determinants of dUTPase temperature optima.