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

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Ribonucleotide reductases (RNRs) are crucial enzymes for DNA synthesis, catalyzing the production of deoxyribonucleotides.
• Their substrate specificity, particularly for noncanonical nucleotides, remains underexplored despite physiological and biotechnological relevance.
• Non-natural nucleotides have significant applications in medicine, biotechnology, and synthetic biology.

Purpose of the Study:

• To investigate the substrate specificity of two thermostable RNRs.
• To determine the substrate promiscuity of RNRs for a wide array of natural and non-natural nucleotides.
• To define the potential and limitations of RNRs in biotechnological applications.

Main Methods:

• Enzymatic assays were performed using two thermostable RNRs.
• A broad range of natural and non-natural nucleotides were tested as substrates.
• Enzyme activity was measured to determine substrate conversion efficiency.

Main Results:

• Both RNRs demonstrated the ability to convert all canonical nucleotides.
• The enzymes successfully processed various non-natural nucleotides, particularly those with modifications to the nucleobase core structure.
• Substrate promiscuity was observed, with a preference for modified functional groups on existing nucleobase structures.

Conclusions:

• Thermostable RNRs exhibit significant substrate promiscuity, accepting a wide range of noncanonical nucleotides.
• The findings highlight the potential for RNRs in biotechnological applications, such as synthetic biology and drug development.
• This study enhances understanding of how RNRs handle naturally occurring nucleotide analogues in cellular metabolism.