Nature Counts to Three: Universal Mg-Pinch Motif Polarizes the Cleaved Bond in NTP-Processing Enzymes

Phosphates are essential for life in all organisms, playing key roles in nucleic acids, signaling, energy transfer, and biosynthesis. We conducted a quantitative analysis of nucleoside triphosphate (NTP) processing enzymes across all enzymatic reactions, revealing their dominance in phosphate reactivity with ATP as the most prevalent substrate. Two main reaction types occur predominantly: cleavage resulting in pyrophosphate release or phosphate release/transfer. The large majority of NTP processing enzymes require divalent Mg²⁺ ions in a mechanistically analogous manner. However, the catalytically competent metal-ion coordination remains unclear for many NTP-processing enzymes. By examining a vast data set of crystallographic structures, we identified a universal "Mg-pinch" motif, confirming our structural hypothesis for almost all NTP processing enzymes. We hypothesized and subsequently confirmed that the catalytic Mg²⁺ typically coordinates the two phosphate groups between which the P-O bond is cleaved. We present a comprehensive analysis of NTP processing superfamilies across all species, determining distinct enzyme active site structures. We highlight exceptional cases and propose challenging superfamilies that lack sufficient structural data to determine precise active site coordination. DFT-based QM/MM calculations with full electrostatic embedding support a mechanistic interpretation in which appropriately coordinated Mg²⁺ ions contribute to catalysis by electrostatic preorganization and polarization of the reacting phosphate groups. The Mg-pinch motif provides a mechanistic framework for understanding the catalytic role of metal ions in NTP processing. Our findings offer insights into enzyme evolution, provide a basis for rational enzyme engineering, and could inform the development of novel therapeutics targeting NTP processing enzymes.