The role of conserved residues in Fdc decarboxylase in prenylated flavin mononucleotide oxidative maturation, cofactor isomerization, and catalysis

Abstract

The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu–Arg–Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277–Arg173–Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu–Arg–Glu network, as only Glu → Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu–Arg–Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu–Arg–Glu network in both catalysis and oxidative maturation.

Exploring the substrate scope of ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae

“…The substrate orientation is further facilitated by R175, interacting with the carboxyl group of the substrate, while the other key residues are E285, acting as acid-base in the reaction mechanism, and E280, which presumably tunes the pKa of R175 and in turn E285 (Fig. 4a) 25 . Accordingly, the reaction rates are influenced by multiple substrate-related factors, such as inductive effects of substituents, presence of extended conjugation, substrate orientation related to the prFMN and within the catalytic site, influenced by both size and planarity of substrate.

Figure 4( a ) Comparison of ligand (α-methyl trans -cinnamate) conformations taken from the 4ZA7 crystal structure (violet) with the lowest energy docking pose (green), and with the reported transition state geometry 24 (orange) of 1,3-dipolar cycloaddition.

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“…S71). The presence of pr FMN cofactor and α-methyl cinnamate in An FDC1 supports an active conformation, compared to the impaired Sc FDC1 structures ( i ) without cofactor 32 (PDB code: 4S13), ( ii ) with the catalytically essential glutamate E285 in inactive conformation 6 (PDB code: 4ZAC) or ( iii ) of mutant E285D (PDB code: 6EVF) with 37 fold decreased k cat value 25 , and ( iv ) with mutation R175A (PDB code: 6EVE), altering hydrogen bonding network implied in substrate fixation and hence inactivating the enzyme 32 . The search space was defined as a cubic box centered at the binding site, with an edge length of 20 Å.…”

Non‐Oxidative Enzymatic (De)Carboxylation of (Hetero)Aromatics and Acrylic Acid Derivatives

“…In order to obtain the catalytically active iminium species of the cofactor (prFMN iminium ), oxidative maturation of reduced prFMN (prFMN reduced ) by molecular oxygen in the presence of apo ‐decarboxylase appears to be crucial [Scheme , (b)] . Although the exact oxidation mechanism of the reduced UbiX product to prFMN iminium is unknown as of yet, a Glu‐Arg‐Glu motif as catalytic acid in the active site of the decarboxylase seems to assist this process . Notably, the prenyl modification in its catalytically active iminium form entirely eliminates the chemical properties of FMN as a redox mediator for hydride transfer and confers a 1,3‐dipolar azomethine ylide and electrophilic iminium ion character, respectively [Scheme , (b)]…”

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

“…Perhaps the difference in decarboxylase activity between the amount of A1905 and S1905 expressed depends on the differences in the prFMN species contained. 25,27 FDC enzymatic activity requires oxygen to form an active prFMN form; however, more oxygen exposure leads to irreversible oxidative maturation to form an inactive form. 28,29 Therefore, in this pathway, anaerobic conditions are suitable for prFMN activation and ccMA production because oxygen is required for catechol ring opening by catA, while anaerobic conditions are needed to maintain FDC activity.…”

“…26 The prFMN-containing FDC can recognize aromatic compounds and α,β-unsaturated carboxylic acids without aromatic rings. 27,28,29,30,31,32,33 FDC can decarboxylate hexadienoic acid, whose structure is very similar to ccMA. 25 Therefore, we hypothesized that by designing a substrate-binding site of FDC to change substrate speci city, FDC could recognize ccMA.…”