Crystallographic studies on the subunits and holocomplex of class Ib ribonucleotide reductase

The enzyme ribonucleotide reductase (RNR) is essential in all cellular organisms since it catalyses the conversion of all four ribonucleotides to corresponding deoxyribonucleotides (dNTPs), the building blocks of DNA. To be able to support the cells with appropriate amounts of dNTPs, RNR is highly allosterically regulated. The biologically active form of class I RNR is thought to be composed of two homodimers, R1 and R2. The enzymatic reduction is initiated by an organic free radical generated by a di-iron site located in the smaller R2 subunit. When the radical is needed for reaction initiation it is transported to the active site in the R1 subunit. This thesis presents structural studies of class Ib RNR from two different pathogenic bacteria. M. tuberculosis codes for two different R2 subunits of the class Ib RNR, R2F-1 and R2F-2 respectively. The crystal structure of the R2F-2 subunit was determined to 2.2 Å. It is an all helical protein with the di-iron site positioned within a four helix bundle. The di-iron site is in its reduced state. Comparison of the R2F-2 structure with a model of R2F-1 suggests that the important differences are located at the C-terminus. The three-dimensional structure of the large subunit of the first member of a class Ib RNR, R1E of S. typhimurium, was determined in its native form and in complex with four of its allosteric specificity effectors. The enzyme contains a characteristic 10-stranded α/β-barrel with catalytic residues at a finger loop in its centre. The N-terminal domain is about 50 residues shorter in the class Ib enzymes compared to the class Ia enzymes, which explains the absence of the allosteric overall activity. The crystal structure of the first holocomplex of any RNR was determined to 4Å. The structure of R1E/R2F from S. typhimurium reveals a non symmetric interaction between the two subunits. There is clear binding of a polypeptide in the hydrophobic pocket of one R1E monomer. The pocket is known to mediate the interaction between the two subunits and we propose that it is the C-terminus of an interacting R2F subunit that binds in the cleft