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

Rebecca Persson

Research output: ThesisDoctoral Thesis (compilation)

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.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biochemistry and Structural Biology
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date1998 Oct 16
Publisher
Publication statusPublished - 1998

Bibliographical note

Defence details

Date: 1998-10-16
Time: 10:15
Place: Sal C, Center for Chemistry and Chemical Engineering, Sölvegatan 39, Lund, Sweden

External reviewer(s)

Name: Nordlund, Pär
Title: Professor
Affiliation: Department of Biochemistry, Stockholm University, Stockholm, Sweden

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Article: dUTPase from Herpes Simplex Virus Type 1; Purification from Infected Green Monkey Kidney (Vero) Cells and from an Overproducing Escherichia coli Strain.Björnberg, O., Bergman, A.-C., Rosengren, A.M., Persson, R., Lehman, I.R. & Nyman, P.O.Protein Expression and Purification (1993) 4:149-159

Article: Crystallization and Preliminary X-Ray Crystallographic Studies of dUTPase from Equine Infectious Anemia Virus.Persson, R., Rosengren, A.M., Nyman, P.O., Dauter, Z. & Cedergren-Zeppezauer, E.S.Protein and Peptide Letters (1997) 4:145-148

Article: Crystal Structure of dUTPase from Equine Infectious Anaemia Virus; Active Site Metal Binding in a Substrate Analogue Complex.Dauter, Z., Persson, R., Rosengren, A.M., Nyman, P.O., Wilson, K.S. & Cedergren, E.S.Accepted for publication by Journal of Molecular Biology

Article: dUTPase from Escherichia coli; High-Level Expression and One-Step Purification.Persson, R., Nord, J., Roth, R. & Nyman, P.O.Submitted to Protein Expression and Purification

Article: dUTPase from Epstein-Barr Virus; Recombinant Expression, Purification and Characterization.Persson, R., Sommer, P., Larsson, G. Zeppezauer, M. & Nyman, P.O.Manuscript

Subject classification (UKÄ)

  • Biological Sciences

Keywords

  • Metabolism
  • dUTP
  • dUTPase
  • nucleotide metabolism
  • retrovirus
  • crystallography
  • herpesvirus
  • Biokemi
  • Biochemistry

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