New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

Abstract

The ubiD/ubiX or the homologous fdc/pad genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone biosynthesis1–3 or microbial biodegradation of aromatic compounds4–6 respectively. Despite biochemical studies on individual gene products, the composition and co-factor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear7–9. We show Fdc is solely responsible for (de)carboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesised by the associated UbiX/Pad10. Atomic resolution crystal structures reveal two distinct isomers of the oxidized cofactor can be observed: an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with drastically altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. While 1,3-dipolar cycloaddition is commonly used in organic chemistry11–12, we propose this presents the first example of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

Regioselective para‐Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase

“…The same effect was observed upon exchange of Arg181 or Glu289, which are located near the carboxylate group of the substrate, to Ala. The analogous Glu282 in Fdc1 is proposed to be required for the donation of a proton to the covalently bound intermediate,14a, 16 which hints at a similar role for Glu289 as a catalytic acid in AroY.…”

“…However, upon purification of the decarboxylase from the E. coli host, only little enzyme activity could be detected for either of the two enzymes, despite additional co‐expression with the UbiD‐associated prenyltransferase UbiX to provide sufficient prFMN in vivo. Upon in vitro reconstitution with reduced prFMN (Figure 1 a),14a, 15 decarboxylation activity could be detected with 3,4‐dihydroxybenzoic acid (3,4‐DHBA, 1 ) following brief exposure to oxygen to generate the active prFMN iminium form (Figure 1 b). The lack of activity for anaerobically reconstituted protein clearly demonstrates the requirement for oxidative maturation of the prFMN cofactor.…”

Terminal Alkenes from Acrylic Acid Derivatives via Non‐Oxidative Enzymatic Decarboxylation by Ferulic Acid Decarboxylases

“…In order to assess the biocatalytic potential of FDCs, three previously described representatives28 from Aspergillus niger (An FDC ), Saccharomyces cerevisae (Sc FDC ) and Candida dubliniensis ( Cd FDC) were each co‐expressed with the native E. coli UbiX in E. coli to produce the holo‐enzymes An FDC UbiX , Sc FDC UbiX and Cd FDC UbiX . In this system, the FDCs were fused with a polyhistidine tag, whereas UbiX was co‐expressed untagged to enable in vivo production of prFMN, allowing for the purification of the prFMN‐bound FDC to homogeneity by Ni affinity chromatography.…”

“…Ferulic acid decarboxylases (FDCs) acting on ′non‐phenolic′ cinnamic acids are an intriguing new class of decarboxylases 27, 28, 29. They are distinct members of the UbiD family of decarboxylases and catalyse the non‐oxidative decarboxylation of acrylic acid derivatives such as cinnamic, ferulic and sorbic acid yielding the corresponding terminal alkenes 30,31.…”

“…Recent structural and mechanistic studies revealed that these enzymes utilise a prenylated derivative of flavin (prFMN), a cofactor synthesised by UbiX 32. FDC‐catalysed decarboxylation of cinnamic acid derivatives mediated by prFMN is proposed to proceed via a 1,3‐dipolar cycloaddition,27,28 in which prFMN acts as 1,3‐dipolar diene owing to its azomethine ylide character 28,33, 34, 35. While this type of transformation – commonly referred to as ′Huisgen‐reaction′36,37 – is widely utilised in heterocyclic synthesis, enzymatic equivalents to this reaction are rare 38, 39, 40, 41…”