Abstract: The prenylated-flavin mononucleotide-dependent decarboxylases (also known as UbiD-like enzymes) are the most recently discovered family of decarboxylases. The modified flavin facilitates the decarboxylation of unsaturated carboxylic acids through a novel mechanism involving 1,3-dipolar cyclo-addition chemistry. UbiD-like enzymes have attracted considerable interest for biocatalysis applications due to their ability to catalyse (de)carboxylation reactions on a broad range of aromatic substrates at otherwise unreactive carbon centres. There are now ～35[thin space (1/6-em)]000 protein sequences annotated as hypothetical UbiD-like enzymes. Sequence similarity network analyses of the UbiD protein family suggests that there are likely dozens of distinct decarboxylase enzymes represented within this family. Furthermore, many of the enzymes so far characterized can decarboxylate a broad range of substrates. Here we describe a strategy to identify potential substrates of UbiD-like enzymes based on detecting enzyme-catalysed solvent deuterium exchange into potential substrates. Using ferulic acid decarboxylase (FDC) as a model system, we tested a diverse range of aromatic and heterocyclic molecules for their ability to undergo enzyme-catalysed H/D exchange in deuterated buffer. We found that FDC catalyses H/D exchange, albeit at generally very low levels, into a wide range of small, aromatic molecules that have little resemblance to its physiological substrate. In contrast, the sub-set of aromatic carboxylic acids that are substrates for FDC-catalysed decarboxylation is much smaller. We discuss the implications of these findings for screening uncharacterized UbiD-like enzymes for novel (de)carboxylase activity.
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With tert.-butylhydroperoxide; diphenyl-phosphinic acid; tetra-(n-butyl)ammonium iodide; In dimethyl sulfoxide; at 90℃; for 8h;Sealed tube;
General procedure: A sealed tube equipped with a magnetic stirring bar was chargedwith substituted (2-azaaryl)methane 1 (0.3 mmol), alpha-amino acid 2(0.45 mmol), Ph 2 PO 2 H (0.15 mmol), TBHP (0.6 mmol), and TBAI (0.06mmol) in DMSO (2 mL). The mixture was stirred under air at 90 °C for8 h. Then the mixture was cooled to r.t. and filtered through a shortsilica gel column (EtOAc). The filtrate was diluted with brine (60 mL)and extracted with EtOAc (2 × 30 mL). The combined organic layerswere dried (MgSO 4 ) and the solvent was removed in vacuo to afford aresidue. The residue was purified by column chromatography (silicagel, petroleum ether/EtOAc) to provide the product.
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