Abstract
The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.
Highlights
The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic andaromatic compounds
His-tagged versions of the VdcC proteins from Bacillus subtilis (BsVdcC) and Sedimentibacter hydroxybenzoicus (ShVdcC; formerly Clostridium hydroxybenzoicum24) were co-expressed with Pseudomonas aeruginosa UbiX in Escherichia coli (BL21 DE3)
The corresponding His-tagged VdcD subunits were expressed in E. coli, and purified VcdD was added to VdcC preparations prior to in vitro prFMN reconstitution
Summary
The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. Recent studies have demonstrated that UbiD enzymes can be used for (hetero)aromatic C–H activation at ambient conditions, providing a route to corresponding acids and derivative compounds[5,6,7]. The UbiD enzyme family is ubiquitous in microbes and is frequently associated with the flavin prenyltransferase, UbiX The latter produces the prFMN cofactor required for the UbiDmediated reversible decarboxylation of unsaturated acids (Supplementary Fig. 1)[11]. Recent protein engineering studies have established Fdc[1] can be evolved to accept carboxylated (hetero)aromatic compounds (i.e. where Cα contributes to the (hetero)aromatic ring), catalytic rates remain low[5]
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