Novel Homologs of Isopentenyl Phosphate Kinase Reveal Class-Wide Substrate Flexibility

The widespread utility of isoprenoids has recently sparked interest in efficient synthesis of isoprene-diphosphate precursors. Current efforts have focused on evaluating two-step isoprenol pathways, which phosphorylate prenyl alcohols using promiscuous kinases/phosphatases. The convergence on isopentenyl phosphate kinases (IPKs) in these schemes has prompted further speculation about the class's utility in synthesizing non-natural isoprenoids. However, the substrate promiscuity of IPKs in general has been largely unexplored. Towards this goal, we report the biochemical characterization of five novel IPKs from Archaea and the assessment of their substrate specificity using 58 alkyl-monophosphates. This study reveals the IPK-catalyzed synthesis of 38 alkyl-diphosphate analogs and discloses broad substrate specificity of IPKs. Further, to demonstrate the biocatalytic utility of IPK-generated alkyl-diphosphates, we also highlight the synthesis of alkyl-l-tryptophan derivatives using coupled IPK-prenyltransferase reactions. These results reveal IPK-catalyzed reactions are compatible with downstream isoprenoid enzymes and further support their development as biocatalytic tools for the synthesis of non-natural isoprenoids.

Automated summary

Recent research has focused on efficient synthesis of isoprene-diphosphate precursors using promiscuous kinases/phosphatases. The study characterized five novel IPKs from Archaea and evaluated their substrate specificity, revealing the synthesis of multiple alkyl-diphosphate analogs. Additionally, the biocatalytic utility of IPK-generated alkyl-diphosphates for the synthesis of non-natural isoprenoids was demonstrated.