Pro1 is a critical catalytic residue in the characterized activities of tautomerase superfamily (TSF) members. Only a handful of members (∼346) lack Pro1 in a sequence similarity network (SSN) that consists of over 11,000 members. Most (294 members) are in the malonate semialdehyde decarboxylase (MSAD)-like subgroup, but the ones characterized thus far have little or no MSAD activity. Moreover, there is little to no activity with other TSF substrates. Five non-Pro1 members were selected randomly for kinetic [using phenylenolpyruvate (PP) and 2-hydroxymuconate (2HM)], mutagenic, inhibition, and crystallographic analysis. Using PP,