Nature counts to three: Universal Mg-pinch motif polarizes the cleaved bond in NTP-processing enzymes

Phosphates are essential for all forms of life, playing key roles in DNA/RNA, signaling, energy storage and transfer, and biosynthetic processes. 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 (i) pyrophosphate or (ii) phosphate release/transfer. The large majority of NTP processing enzymes require divalent Mg2+ ions in a mechanistically analogous manner. Despite their importance, the precise coordination of metal ions in the catalytically competent active site structure in many NTP processing enzymes remains elusive. We hypothesized that efficient phosphate processing requires specific metal ion coordination, facilitating the catalytic reaction. By examining a vast dataset of crystallographic structures, we identified a universal "Mg-pinch" motif, confirming our structural hypothesis for almost all NTP processing enzymes. We postulated that the Mg2+ ion should coordinate both phosphates that are involved in the cleaved P-O bond. 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. Our quantum mechanical calculations based on DFT-based QM/MM with full electrostatic embedding revealed the essential electrostatic polarization effects of the Mg²⁺ ion as key determinants of their role in the enzyme catalysis. 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.