AbstractPolyphosphate kinases (PPKs) are valuable biocatalysts for ATP cofactor regeneration as well as for the phosphorylation of non‐canonical nucleotides. The versatility of PPKs in the latter application is defined by their substrate scope. In this study, we investigate the substrate spectrum of the PPK2‐III enzyme from Meiothermus ruber (MrPPK) for the conversion of canonical and non‐canonical (deoxy)‐ribonucleotides. The enzyme shows high substrate promiscuity for purine and to minor degree pyrimidine substrates, with an overall preference for the native substrate AMP. With structure guided rational amino acid exchanges in the active site, we produced an MrPPK variant with improved activity for a broad variety of purine nucleotides. While the preference for AMP is lost, conversion of nucleotides without a 6‐amino function at the purine moiety is increased. This MrPPK variant is a versatile biocatalyst for the synthesis of non‐canonical nucleotides and could also be useful as a GTP cofactor regeneration system.