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The research at Durham University has identified a novel chemo-enzymatic approach to nucleoside-5'-triphosphates (NTPs) synthesis.
About
The existing methods of NTP synthesis rely on either biology or synthetic chemistry and are characterised by several drawbacks that include technically challenging procedures and use of expensive reagents. The technology developed by researchers from Durham University is based on using a mutant version of nucleoside diphosphate kinase (NDPK), an enzyme involved in NTP synthesis. NDPK works by transferring a phosphoryl group from a triphosphate donor to a nucleoside diphosphate substrate. It has been previously shown that wild type NDPK fulfils its function by forming a phospho-histidine intermediate.1 Although a His->Gly mutant of NDPK from Dictyostelium discoideum lacks this function, its enzymatic activity could be restored by adding imidazole.2 The research team at Durham University has deduced that by using the reverse process, H122G NDPK could be used to produce NTPs while using NDPs as substrates and IM-P as the phosphate donor. Phosphoryl imidazole (Im-P) was shown to act as an efficient substrate for the H122G mutant NDPK. Using Im-P allowed for the enzyme activity to be restored as Im-P functions as a replacement for the mutagenized active site histidine residue and delivers a phosphate group.
Key Benefits
• The discovery provides an opportunity to manufacture nucleotides in a cost-efficient manner via chemo-enzymatic approach. • The starting materials used can be conveniently prepared from inexpensive raw materials. o The substrate used in the proposed approach, Im-P, is prepared from phosphoryl chloride (<£10 per mol) and imidazole (<£12 per mol) under simple aqueous conditions. The resulting material can be used crude or isolated via extraction and crystallization procedures. • Much simpler process of NTP synthesis via chemo-enzymatic route compared with the current chemical synthesis approach • The method can be implemented in a various of biotechnological settings
Applications
• Nucleic acid amplification methods incl. PCR • Nucleic acid synthesis • Therapeutics e.g. preparation of siRNAs by in vitro transcription methods • Diagnostics • DNA sequencing • Flow systems that would feed the produced NTPs into NTP-consuming systems