Negative Cooperativity in the Mechanism of Prenylated-Flavin-Dependent Ferulic Acid Decarboxylase: A Proposal for a “Two-Stroke” Decarboxylation Cycle

Abstract

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.

Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase

“…Recent studies on UbiD-like enzymes have suggested the importance of domain motions in catalysis. For example, stopped-flow spectroscopy indicates that the dimeric UbiD-like enzyme FDC exhibits negative cooperativity between the two subunits arising from a conformational change that facilitates inter-conversion between the “fast, tight” and the “slow, loose” active sites ( 46 ). Studies on vanillic acid decarboxylase suggest that “open” and “closed” conformers of the enzyme have different affinities for reaction intermediates that may be important for catalysis ( 44 ).…”

An Enzyme Catalyzing the Oxidative Maturation of Reduced Prenylated-FMN to Form the Active Coenzyme

“…The UbiD pfam of putative prFMN-dependent (de)­carboxylases now contains over 35 000 sequences, pointing to the widespread occurrence of this class of enzymes in microbes. There has been a significant expansion of our knowledge surrounding the mechanisms, − structures, − and substrate range ,,, of these enzymes since the first prFMN-dependent enzyme, FDC, was described in 2015 . However, the biosynthesis of the prFMN cofactor has received much less attention.…”