Negative Cooperativity in the Mechanism of Prenylated-Flavin-Dependent Ferulic Acid Decarboxylase: A Proposal for a...

Ferulic acid decarboxylase (FDC) catalyzes the reversible carboxylation of various substituted phenylacrylic acids to produce the correspondingly substituted styrenes and CO₂. FDC is a member of the UbiD family of enzymes that use prenylated-FMN (prFMN) to catalyze decarboxylation reactions on aromatic rings and C-C double bonds. Although a growing number of prFMN-dependent enzymes have been identified, the mechanism of the reaction remains poorly understood. Here, we present a detailed pre-steady-state kinetic analysis of the FDC-catalyzed reaction of prFMN with both styrene and phenylacrylic acid. Based on the pattern of reactivity observed, we propose a "two-stroke" kinetic model in which negative cooperativity between the two subunits of the FDC homodimer plays an important and previously unrecognized role in catalysis. In this model, catalysis is initiated at the high-affinity active site, which reacts with phenylacrylate to yield, after decarboxylation, the covalently bound styrene-prFMN cycloadduct. In the second stage of the catalytic cycle, binding of the second substrate molecule to the low-affinity active site drives a conformational switch that interconverts the high-affinity and low-affinity active sites. This switching of affinity couples the energetically unfavorable cycloelimination of styrene from the first site with the energetically favorable cycloaddition and decarboxylation of phenylacrylate at the second site. We note as a caveat that, at this point, the complexity of the FDC kinetics leaves open other mechanistic interpretations and that further experiments will be needed to more firmly establish or refute our proposal.