Continuous UV-Spectroscopic Interrogation of Enzymes Processing Phosphorylated Intermediates through Modular Reaction Networks

Phosphorylated intermediates are ubiquitous across primary and secondary metabolism, yet continuous and versatile assay systems for the interrogation of the enzymes that use them are underdeveloped. To address this gap, we developed a system for Phosphate detection by Sucrose phosphorolysis, Isomerization and oxidation, the PSI (or Ψ) module. In contrast to existing assays, Ψ provides a continuous UV-based read-out at neutral pH in different buffer systems and can readily be incorporated into longer reaction sequences to assay a variety of different enzymes of interest. We herein describe the development of Ψ and demonstrate the utility of an optimized variant for the interrogation of prenyltransferases, isomerases, and kinases.