On Monomeric and Trimeric dUTPases. Recombinant Expression, Purification, Conformational Properties and Catalytic Characteristics

Abstract: Nucleotide metabolism and replication of nucleic acids are processes of fundamental importance. A crucial enzyme in nucleotide metabolism is dUTPase (E.C. 3.6.1.23), that catalyses the hydrolysis of dUTP to dUMP and pyrophosphate. The activity of the enzyme has influence on the outcome of DNA replication and complete loss of enzyme activity is lethal, while a decreased activity promotes increased occurance of uracil in DNA, leading to DNA damage and mutations. Many biological systems, including prokaryotes, eukaryotes and several viruses, encode dUTPase activity. The existence of virally encoded dUTPases emphasises the biological importance of the enzyme and many viruses have been demonstrated to depend on their dUTPase activity for maintenance of optimal virulence, thereby providing means for using the viral dUTPases as targets for anti-viral inhibitors. The crystal structures of the homotrimeric dUTPase from the retrovirus equine infectious anaemia virus (EIAV) have been determined for unliganded and liganded forms of the enzyme. The presence of the substrate analogue and inhibitor dUDP in the structure of the complexed enzyme allowed for identification of the active sites (three per enzyme molecule), positioned at subunit interfaces. Four of the five conserved motifs present in most dUTPases are visible in the structure and they are all located in the active site. The fifth (and invisible) motif is presumed to be contributed by the third subunit and to cover the active site during catalysis. A strontium ion, present in the structure of the complexed enzyme, and located in the active site, indicates the position of the metal ion shown to be essential for efficient catalysis. The mammalian herpes virus dUTPases have a molecular organisation differing from those of prokaryotes, eukaryotes and retroviruses, as they are monomeric and generally of increased size. Additionally, the order of the conserved motifs is rearranged. Protocols for recombinant expression and purification of dUTPases from two herpes viruses (HSV-1 and Epstein-Barr virus (EBV)) have been established. The activity and solubility of both enzymes were shown to be influenced by presence of detergent. A kinetic characterisation of the enzyme from Epstein-Barr virus has been initiated and shows the enzyme to have properties similar to the HSV-1 enzyme. Using a stopped-flow technique the KM value of the EBV enzyme was determined to 0.2 micromolar. The dut gene encoding E. coli dUTPase has been recloned and a simplified purification procedure designed, allowing for recovery of 500 mg pure enzyme per litre of bacterial culture.