Ferulic acid decarboxylases (Fdc) are decarboxylase

enzymes Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...

capable of the reversible

decarboxylation Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain. The reverse process, which is t ...

of aromatic carboxylic acids such as ferulic acid and cinnamic acid. Fdc's are fungal homologues of the '' E.coli'' UbiD enzyme which is involved in ubiquinone biosynthesis. This places Fdc within the wider UbiD enzyme family, representing a distinct clade within the family Presence of ''fdc1'' and the associated ''pad1'' genes (Pad1 homologous to UbiX in ''E.coli'') were shown to be required for the decarboxylation of phenylacrylic acids in ''Saccharomyces cerevisiae''. In 2015 the cofactor prFMN was discovered in the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...

of Fdc1 from '' Aspergillus niger'' (AnFdc) by

crystallography Crystallography is the experimental science of determining the arrangement of atoms in crystalline solids. Crystallography is a fundamental subject in the fields of materials science and solid-state physics (condensed matter physics). The wor ...

, prior to this genetic studies had led to the assumption that both UbiD and UbiX encoded isofunctional decarboxylases. In actuality UbiX/Pad were found to be flavin preyltransferases supplying the prFMN cofactor to UbiD/Fdc where it is utilised for the reversible decarboxylation of alpha-beta unsaturated carboxylic acid substrates. Since the discovery of prFMN AnFDC has become the most well understood representative of the UbiD enzyme family

AnFDC Mechanism

In the same paper in which the structure of prFMN was deduced in the active site of AnFdc1 there was a proposal for the mechanism by which Fdc1 decarboxylates α,β-unsaturated carboxylic acids. Not all UbiD enzymes decarboxylate

acrylic acid Acrylic acid (IUPAC: propenoic acid) is an organic compound with the formula CH2=CHCOOH. It is the simplest unsaturated carboxylic acid, consisting of a vinyl group connected directly to a carboxylic acid terminus. This colorless liquid has a ...

substrates and other mechanisms may be at play for alternative substrates. In the case of AnFdc1 it was noted that prFMN displays an azomethine ylide characteristic C4a-N5+=C1’(Figure 1). This is a well-known

1,3-dipole In organic chemistry, a 1,3-dipolar compound or 1,3-dipole is a dipolar compound with delocalized electrons and a separation of charge over three atoms. They are reactants in 1,3-dipolar cycloadditions. The dipole has at least one resonance st ...

in organic chemistry, positioned in the enzyme active site near to the α,β-unsaturated carboxylic acid substrate which contains a 1,3-dipolarophile. Thus, it was proposed that a 1,3-dipolar cycloaddition mechanism was responsible for the enzymatic decarboxylation. This was confirmed in a later paper. The mechanism proposed in for 1,3-dipolar cycloaddition by Fdc1 is as follows (intermediates represented in Figure 1): # 1,3-dipolar cycloaddition between prFMN^iminium and the α,β-unsaturated substrate leads to a

pyrrolidine Pyrrolidine, also known as tetrahydropyrrole, is an organic compound with the molecular formula (CH2)4NH. It is a cyclic secondary amine, also classified as a saturated heterocycle. It is a colourless liquid that is miscible with water and most ...

cycloadduct (Int1) # This pyrrolidine cycloadduct supports simultaneous decarboxylation and ring opening, resulting in the formation of a distinct prFMN-alkene adduct (Int2) # A conserved

glutamic acid Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can synt ...

residue (E282) donates a proton to the alkene moiety, resulting a second pyrrolidine cycloadduct (Int3) # The reaction concludes with cycloelimination of Int3 and the release of the alkene product and CO₂ A study went on to present evidence for the 1,3-dipolar cycloaddition, due to suspected turnover of cinnamic acid a crystal structure of AnFdc1 in complex with α-fluorocinnamic acid revealed the substrate Cα and Cβ carbons are located directly above the prFMN^iminium C1’ and C4a respectively (shown as Sub in Figure 1 - with cinnamic acid as opposed to α-fluorocinnamic acid). Cinnamic acid was confirmed to bind in a similar manner using inactive AnFdc1 crystals containing FMN. The AnFdc1 E282Q mutant crystallised with cinnamic acid revealed a structure corresponding to the Int2 species, this was taken to mean that progression through the 1,3-dipolarcycloadition cycle was halted as E282 is unable to donate a proton to the alkene moiety. In order to observe the Int1 and Int3 structures

alkyne \ce \ce Acetylene \ce \ce \ce Propyne \ce \ce \ce \ce 1-Butyne In organic chemistry, an alkyne is an unsaturated hydrocarbon containing at least one carbon—carbon triple bond. The simplest acyclic alkynes with only one triple bond and n ...

analogues were used. Like alkenes these compounds can also act as dipolarophiles but cycloaddition would yield a cycloadduct containing a double bond. An inactive AnFdc1 enzyme (with prFMN^radical bound) co-crystallised with the phenylpropiolic acid revealed binding in a similar manner to the α-fluorocinnamic acid AnFdc1 and cinnamic acid AnFdc1 with FMN bound (Inhib). An active AnFdc1 enzyme co-crystallised with phenylpropiolic acid revealed clear density for a 3-pyrroline cycloadduct (Int3’) between the alkyne and prFMN^iminium. Int3’ represents a structure post decarboxylation, so it was assumed that over the time it took for crystallisation (~24h) the decarboxylation had occurred. Using a rapid soaking procedure, a different cycloadduct was observed that retained the carboxyl moiety (Int1’).

References

{{reflist, 30em Enzymes Acids Genetics Biochemistry