Conversion of Inactive Non-Pro1 Tautomerase Superfamily Members into Active Tautomerases: Analysis of the Pro1 Mutants

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, kcat/Km values (∼101-102 M-1 s-1) could be estimated for three native proteins whereas using 2HM, a kcat/Km value could only be estimated for one native protein (∼103 M-1 s-1). The kcat and Km values could not be determined. However, changing the N-terminal residue to a proline gave a significant improvement in kcat/Km values for all mutant enzymes using PP or 2HM. For PP, the kcat/Km values ranged from 103-105 M-1 s-1 and for 2HM, the kcat/Km values ranged from 102-104 M-1 s-1. In addition, it was now possible to measure kcat and Km values for all mutant proteins using PP and one mutant protein using 2HM. Incubation of the Pro1 mutants with 3-bromopropiolate (3BP) results in covalent modification of the prolyl nitrogen of Pro1 by a 3-oxopropanoate adduct. Crystallographic analysis of two mutant enzymes (NJ7V1P and 8U6S1P) modified by the 3-oxopropanoate adduct identified binding ligands and suggest a mechanism for the tautomerase activity involving Pro1, Arg71, Tyr124, and the backbone amide of Phe68.