Mechanistic Studies on the Prenylated- Flavin-Dependent Phenazine-1-Carboxylic Acid Decarboxylase

Abstract

Decarboxylases are chemically versatile enzymes capable of manipulating C–C bonds by reversibly converting carboxylic acids to their corresponding hydrocarbons. Thus, they are being considered as viable biocatalysts for the sustainable production of commodity chemicals. The formation of a carbanion intermediate poses a significant kinetic barrier to decarboxylation and therefore, nature has evolved cofactors such as thiamine pyrophosphate (TPP), pyridoxal-5’-phosphate (PLP) as well as metal ions, to facilitate the reaction.

Recently, a modified flavin cofactor was discovered that contains an extra 6 membered ring between the N5 and C6 positions of the isoalloxazine moiety. Named as prenylated flavin mononucleotide (prFMN), this cofactor features a unique azomethine ylide that is essential for catalysis. The UbiD-family of decarboxylases, named after the archetypical enzyme found in bacterial ubiquinone biosynthesis, utilizes prFMN to (de)carboxylate a number of α,β-unsaturated, (hetero)aromatic and phenolic carboxylic acids. In the well-studied enzyme ferulic acid decarboxylase (FDC), the reaction proceeds through the formation of a 1,3-dipolar cycloadduct between prFMN and the substrate, trans-cinnamic acid. On the other hand, in the protocatechuic acid decarboxylase AroY, an electrophilic mechanism is suggested. Overall, a detailed characterization of different UbiD-like enzymes can uncover novel mechanisms and benefit their development as biocatalysts. While FDC has been studied extensively, a kinetic evaluation of other UbiD-like enzymes is lacking. These enzymes are known to crystallize in distinct ‘open’ and ‘closed’ conformers but their relevance to catalysis also remains to be discovered. Lastly, the biggest hurdle in studying UbiD-like enzymes is that oxidative maturation of prFMN is poorly understood. My work addresses some of these problems in the field of UbiD-catalyzed reactions.

Initially, I characterized a recently discovered prFMN dependent enzyme from Mycolicibacterium fortuitum. Named PhdA, this enzyme decarboxylates phenazine-1-carboxylic acid, providing M. fortuitum a competitive advantage over phenazine producers in soil. I developed an optimal method for reconstituting PhdA that doesn’t require the use of reducing agents described previously. Moreover, I showed that PhdA can decarboxylate a number of (hetero)aromatic carboxylic acids, including anthracene-1-carboxylic acid. It also catalyzes the much slower exchange of solvent deuterium in phenazine. Finally I proposed a 1,3-dipolar cycloaddition mechanism for PhdA.

For a detailed analysis of PhdA’s mechanism, I studied solvent isotope and viscosity effects. Surprisingly, I discovered that D2O-associated changes in protein conformations significantly improved reaction rates. Molecular dynamics (MD) simulations performed in collaboration with Soumil Joshi and Dr. Sanket Deshmukh from Virginia Tech suggest that D2O leads to domain closure, akin to the ‘closed’ conformer observed in crystal structures of several UbiD-like enzymes. Given that many UbiD-like enzymes crystallize in the ‘open’ form, these results show that optimizing solvent systems and/or engineering to adapt a ‘closed’ conformer might improve the efficiency of UbiD-catalyzed reactions.

Finally, I studied the biosynthesis and maturation of prFMN in detail to shed light on this process. I showed that the in vitro prenylation of FMN catalyzed by UbiX is inefficient and several products are formed that affect prFMN maturation. These species (collectively called as prFMNox) subsequently undergo solvolysis to re-form FMN as well as other degradation products.

Overall, my work shed light on the yet poorly understood prFMN maturation, expanded the substrate scope of UbiD-like enzymes and identified a novel way to engineer these proteins. This research would benefit future studies, improving scientific scholarship and building towards a more sustainable way for synthesizing commodity chemicals.