Abstract
Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an “open-closed” mechanism involving a pocket of 8×8×15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer.
Highlights
Phenolic acids, mainly p-coumaric and ferulic acids, are covalently bound to polysaccharides in cell walls of higher plants
The crystal structures were determined to 2.4 Aand 2.1 Aresolutions respectively for the pure ferulic acid decarboxylase (FADase) and FADase complexed with sodium ferulate
A gene encoding the FADase enzyme was isolated from Enterobacter sp
Summary
Mainly p-coumaric and ferulic acids, are covalently bound to polysaccharides in cell walls of higher plants These acids are essential for the growth and reproduction of plants, and are commonly produced as part of the defense against pathogen infection at injured sites in plants [1]. Ferulic acid [3-(4hydroxy-3-methoxyphenyl)-2-propenoic acid] is an abundant hydroxycinnamic acid in the plants and can be transformed by microorganisms into valuable aromatic compounds such as vinylguaiacol and vanillin [2]. In plants, this acid may exist in a free form or be covalently linked to lignin and other polymers in the cell wall [3]. Strong market demand for natural vanillin has spawned efforts to produce it by microbial transformation from natural substrates, including phenolic stibenes [8], eugenol [9,10], and ferulic acid [7,11]
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