Substrate Specificity of B12-Depedent Ribonucleotide Reductases: Biotechnology and Metabolic Implications

Ribonucleotide reductases (RNRs) catalyze one of the central biochemical reactions, giving rise to deoxyribonucleotides, the building blocks of DNA. Due to their importance in cellular metabolism, this class of enzymes has been extensively studied over five decades. One aspect that has been neglected so far is the substrate specificity in terms of noncanonical nucleotides. While some of these compounds are physiologically relevant, many non-natural nucleotides are important in medical science, biotechnology and synthetic biology. In this study, we investigated the substrate specificity of two thermostable RNRs for a broad range of natural and non-natural nucleotides, in order to define the substrate promiscuity of this class of enzymes. Both enzymes were capable of converting all canonical nucleotides and a variety of other nucleotides. Generally, the enzymes were more likely to convert substrates with modifications of already existing functional groups of the nucleobase core structure. Our results show the potential and limitations for the biotechnological application of RNRs. In addition, they improve our understanding of the natural nucleotide metabolism in dealing with naturally occurring nucleotide analogues.