Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily

-fold difference in native specificities appears to arise from distinct interactions at a single phosphoryl substituent. We also uncovered striking mechanistic similarities between the PafA and EcAP monoesterases, including evidence for ground-state destabilization and functional active-site networks that involve different active-site groups but may play analogous catalytic roles. Discovering common network functions may reveal active-site architectural connections that are critical for function, and identifying regions of functional modularity may facilitate the design of new enzymes from existing promiscuous templates. More generally, comparative enzymology and analysis of catalytic promiscuity can provide mechanistic and evolutionary insights.