• Derek Logan

Ribonucleotide reductases (RNRs) are essential enzymes for all life by virtue of their central role in the de novo synthesis of deoxyribonucleotides, the building blocks for DNA synthesis. Organisms possess dNTP salvage pathways, but RNRs are responsible for the last and essential step of synthesis "from scratch". RNRs have fascinated a wide range of researchers, from geneticists through biochemists and biophysicists to theoretical chemists, since Peter Reichard discovered them in the 1950s. This can be attributed to their diversity and to their use of protein-based radicals. In addition, they have among the most complex allosteric regulations of any enzyme system. Activity is regulated on two levels: overall activity and substrate specificity (as only one RNR catalyses the reduction of all four ribonucleotides under given physiological conditions). dNTP products regulate the enzyme's specificity for the NTP or NDP substrates.

RNRs can be divided into three classes based on their mechanisms of radical generation. Class I enzymes are totally O₂-dependent and generate a stable tyrosyl radical on the protein through activation of O₂ by a di-iron centre. Class II RNRs generate a transient 5'- deoxyadenosyl radical through cleavage of the C-Co bond in adenosylcobalamin. They can function either in the presence or in the absence of O₂. Class III RNRs are strictly anaerobic and generate a stable glycyl radical on the protein by cleavage of S- adenosylmethionine, which can be regarded as a homologue of adenosylcobalamin. It is thought that all of these radicals are transferred to cysteine side chains in order to initiate the reaction.

We are particularly interested in the structure and function of class II and III RNRs. In 1999 we solved the structure of the anaerobic RNR from bacteriophage T4, pictured left. This was the first structural proof of a common evolutionary origin for the three classes of RNR and also pointed to an evolutionary relationship to the glycyl radical enzyme pyruvate formate lyase (PFL). More recently we have probed the allosteric regulation of class II and class III RNRs by solving the structures of complexes with several combinations of allosteric effector and substrate [2,5]. We have been able to explain how conformational changes a loop spanning the dimer interface communicate the substrate specificity signal to the active site for the class II RNR. Similar movements were observed for the class III enzyme but the full picture has still not been obtained as substrates were not observed to bind to the active site in this system.

Work in progress:

We continue to try to obtain substrate binding to the active site of class III RNRs. Recently we discovered a novel metal centre in the C-terminus of the enzyme [3] which we are investigating using various methods. We are also determining the structure of other class III RNRs which have overall activity regulation (the T4 enzyme does not), as well as of mutants of the T4 RNR which affect the catalytic mechanism and the allosteric regulation.

Collaborators:

This work is a collaboration with the groups of Prof. Britt-Marie Sjöberg, Stockholm University, Prof. Marc Fontecave & Dr. Etienne Mulliez, CEA/CNRS, Grenoble, Dr. David Edgell, University of Western Ontario, London, Canada.