Kinetic and structural characterization of human NUDIX hydrolase NUDT15 and NUDT18 as catalysts of isoprene pyrophosphate hydrolysis

Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules, and serve as substrates for post-translational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been reported to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been identified. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human NUDT15 and NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, which displays a catalytic efficiency of 2.1x10^5 M-1s-1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0x10^4 M-1s-1. LC-MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 A. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and aided in identifying key binding residues. By overexpressing NUDT15 and NUDT18 in cells, we demonstrated a decrease in cellular cholesterol levels. Collectively, our findings strongly imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, and support their involvement in animal isoprene pyrophosphate metabolism. ### Competing Interest Statement The authors have declared no competing interest.