The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp catalytic triad to hydrolyze a C-C bond. DxnB2 is a HOPDA hydrolase that catalyzes the C-C bond hydrolysis of the biphenyl metabolite, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). The hydrolysis of HOPDA has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ESred, which possesses a marked bathochromically shifted absorption spectrum. Here, the authors investigated the catalytic mechanism of MCP hydrolases using HOPDA hydrolase DxnB2 of Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of HOPDA to 2-hydroxy-2,4-pentadienoic acid and benzoate supported a nucleophilic mechanism catalysis. In DxnB2, the rate of ESred decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limited the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on the MCP pKa2 value (βnuc = ∼1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic His residue was displaced upon substrate-binding. The results provided evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggested that ESred represents a dianionic intermediate that acts as a general base to activate the Ser nucleophile. Thus, this substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases. [on SciFinder(R)]