To reduce a ribonucleotide – Radical solutions in enzymology in form and function

Detta är en avhandling från Department of Biochemistry and Structural Biology, Faculty of Science, Lund University

Sammanfattning: It has been more than 50 years since the enzyme system ribonucleotide reductase (RNR), catalysing the reduction of ribonucleotides to deoxyribonucleotides, was first discovered. RNR was also the first time that radical chemistry was revealed in an enzyme. RNRs carry out a key step in the de novo synthesis of building blocks for DNA and have been found in almost all known organisms. Within this thesis the anaerobic RNR from the thermophilic bacteria Thermotoga maritima has been studied by making use of X-ray crystallography, anaerobic enzyme assays, small-angle X-ray scattering and complementary biophysical methods. The crystal structure of the catalytic subunit of this anaerobic RNR stood out by lacking the typical cysteine residue, which takes part in radical transfer to the substrate, positioned in its active site. The opposing site for the glycyl radical was however structurally conserved, as in structures of other glycyl radical enzymes. It was shown that the glycyl radical could be generated by the addition of the radical generating subunit with a reduced Fe4S4 cluster and the co-substrate S-adenosyl methionine. By also adding T. maritima cell extracts it was verified that the system was an active RNR. Further work focused on understanding complex formation between the two subunits and the structural manifestation of allosteric regulation. Anaerobic analytical ultracentrifugation indicated that a dimer of the catalytic subunit interacts with a monomer of the radical generating subunit. Crystal structures of the catalytic subunit in complex with various nucleotides were used to compare hydrogen bonding networks and nucleotide recognition for allosteric regulation. The second model system studied was the Pseudomonas aeruginosa aerobic RNR catalytic subunit. Proteins from P. aeruginosa are of clinical interest as possible drug targets, due to the many human infections caused by this microorganism. By solving the crystal structure of the catalytic subunit in complex with dATP it became possible to visualise its tetramerisation interface and its surprising capacity to bind three dATP molecules over two ATP cones per monomer. This work shows how RNRs, present in very different types of organisms, still keep on delivering surprises and different solutions to the complexity of synthesising deoxyribonucleotides and regulating nucleotide pools in the cells.

  Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.