Structure-guided engineering of a polyphosphate kinase 2 class III from an Erysipelotrichaceae bacterium to produce base-modified purine nucleotides.

Nucleobase-modified nucleoside-5'-triphosphates (NTPs) are important building blocks for the enzymatic synthesis of non-coding RNAs and mRNAs with improved properties. Chemical phosphorylation of base-modified nucleotides to NTPs remains challenging. Here, we report the enzymatic phosphorylation of purine-modified nucleoside-5'-monophosphates (NMPs) to the corresponding NTPs by the polyphosphate kinase 2 class III from an Erysipelotrichaceae bacterium (EbPPK2). The enzyme is highly promiscuous, accepting a range of NMPs with purine modifications. EbPPK2 efficiently catalyses the formation of the corresponding di-, tri- and tetraphosphates, typically with >70% conversion to the NTP. Slower conversion was observed for analogues with oxo- or thio-substitutions at the C6-position. To better understand nucleotide binding and catalysis, we determined the crystal structure of EbPPK2 at 1.7 Ã resolution bound to a non-hydrolysable ATP analogue and polyphosphate. This enabled structure-guided design of EbPPK2 variants that efficiently convert GMP analogues, while retaining activity for AMP. Apart from being the preferred industrial-scale ATP recycling catalyst, EbPPK2 and variants bear potential to become the favoured enzyme family for purine-modified NTP production.