Construction of a ribonucleotide reductase R1 protein with an active site selenocysteine

The components of DNA, the deoxyribonucleotides, are produced from the components of RNA, the ribonucleotides. One single substitution is needed to convert a ribonucleotide into a deoxyribonucleotide i.e. a replacement of a hydroxyl group with a hydrogen atom. The reaction is catalysed by ribonucleotide reductase, an enzyme that is present in all living organisms. At first this conversion may seem trivial but in reality it is a difficult chemical reaction requiring much energy. In ribonucleotide reductase this energy is provided by an amino acid radical that upon each catalytic turnover is transferred from its stable position in the R2 protein to the active site of protein R1. The need for deoxyribonucleotides in the cell varies, therefore the activity of ribonucleotide reductase must be regulated. A complex allosteric regulation controls both the level of enzymatic activity and the substrate specificity to make sure that the deoxyribonucleotides are produced in correct amounts. In this work, we have shown that the reason why enzymatic activity is turned off when dATP binds is due to formation of a constrained R1: R2 interaction and we have proposed that a conserved hydrogen bond is important in this mechanism. We evaluated the effect of nucleotides on the R1: R2 interaction further using the surface plasmon resonance technique and found that allosteric effectors and substrates as well as the presence of thioredoxin considerably enhances the interaction.The second allosteric site that controls substrate specificity is located at the interaction area of the two polypeptides constituting protein R1. We have identified key residues in the dimerisation process of the two polypeptides and we have established a stabilisation effect of allosteric effectors and substrates on the dimer interaction using a mutant protein. The radical is believed to be transferred between the two proteins that constitute ribonucleotide reductase by a chain of hydrogen bonded amino acid residues. We have developed a new in vivo activity assay in which we have shown the physiological importance of these residues. Another methodological approach in this work was an attempt to turn protein R1 of ribonucleotide reductase into a selenoprotein by genetically substituting one of the active site cysteines for a selenocysteine. In a selenocysteine-substituted protein, this particular residue can be distinguished from the other cysteines which would be advantageous in subsequent biophysical characterisations of thiyl radicals.