Ribonucleotide reductase (RNR) catalyse the conversion of ribonucleotides to their corresponding deoxyribonucleotides in all organisms. The deoxyribonucleotides are the building blocks for DNA. Three different classes of RNR are found, class I, II and III. The class I RNRs operate under aerobic conditions, the class III RNRs operate under anaerobic conditions and the class II RNRs are indifferent to oxygen. All classes of RNR catalyse the reaction using a free radical mechanism. The free radical is generated to initiate the reaction mechanism but the generation differs between the classes.
I have worked with the anaerobic class III RNR from bacteriophage T4 and the work presented in this thesis involves several different aspects of the enzyme. The class III RNR from phage T4 can be used as a model for other class III RNRs.
From isotope labelling experiments, we show that a stable glycyl radical forms in the phage T4 class III RNR. I used site-directed mutagenesis to locate the glycyl radical to Gly580 in the NrdD protein of the T4 class III RNR. The glycyl radical is absolutely required for enzymatic activity.
Also using protein engineering, I show for the first time, the importance of cysteines in radical generation and the reaction mechanism of the class III RNRs. Four cysteines in the C-terminal of T4 NrdD are responsible for the last step in the generation of the glycyl radical at Gly580. Two cysteines in the active site of T4 NrdD, Cys79 and Cys290 are required for the reaction mechanism of the enzyme. A third residue within the active site, Asn311 is most likely also important for catalytic activity. A reaction mechanism that is different from the class I and II RNRs has been proposed.
The first crystal structure of a class III RNR, the class III RNR from phage T4 is presented. Structural relationships with the known class I RNR structure is discussed as well as similarities with another glycyl-radical enzyme.
Finally, the allosteric regulation of the class III RNR from phage T4 was characterized. Almost all RNRs are allosterically regulated to keep the deoxynucleotide pools balanced in the cell. Similarities to other RNRs as well as a unique feature of the class III RNR from phage T4 is discussed.
2000. , 67 p.