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Multiple Catalytic Promiscuity: Specificity and Efficiency in the Alkaline Phosphatase Superfamily

‘Promiscuous’ enzymes possess additional activities in addition to their native ones, challenging the textbook adage “one enzyme – one activity”. In particular the question arises, whether efficiency and specialisation are inextricably linked. The observation of strong promiscuous activities in the alkaline phosphatase (AP) superfamily ‐ where one active site can catalyse up to six chemically distinct hydrolytic reactions with promiscuous second order rate accelerations between 109 and 1017‐ suggests that even broadly promiscuous catalysis can be rather efficient. Mechanistic and structural studies are used to delineate how efficiency and specificity can be combined and managed. We demonstrate that crosswise promiscuity relationships in the AP superfamily indicate that an enzyme is ‘pregnant’ with another activity, i.e. has the potential to be mutated or evolved into a new catalyst. These catalysts are multifunctional generalists that have won additional activities, at low trade‐off cost to the other existing activities. In particular, we use picoliter water‐in–oil emulsion droplets produced in microfluidic devices as high‐throughput screening reactors and present a workflow that allows screening of >;106 clones and allows successful selections, where lower throughput approaches have failed.