A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (±-t6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30"C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2-or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2', Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.Ferulic acid (compound 1 in Fig. 1) is an extremely abundant, lignin-related aromatic acid of interest as a renewable resource for the production of useful aromatic chemicals. We are exploiting the use of enzymes and microbial transformations as a means of generating value-added products from ferulic acid (17,18). An understanding of the biochemical and enzymatic processes involved in ferulic acid biotransformations is required as a theoretical basis for the ultimate development of biocatalytic processes for the production of large amounts of ferulic acid-derived aromatic chemicals. Whole-cell bioconversions of ferulic acid to 4-hydroxy-3-methoxystyrene (compound 3 in Fig. 1) have been reported to occur in bacteria and fungi (1,2,17,18,(23)(24)(25). Some properties of the enzymes decarboxylating 4-hydroxycinnamic acid have been studied with crude cell extracts from an Aerobacter sp. (11) and with a partially purified enzyme from Cladosporium phlei (13). Surprisingly, no work on the purification and characterization of the enzyme(s) catalyzing the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene has been reported to date.We have identified a strain of Pseudomonas fluorescens (UI-670) that efficiently transforms ferulic acid (compound 1) to 4-hydroxy-3-methoxystyrene (compound 3) (Fig. 1). Deuterium labeling experiments confirmed the intermediacy of compound 2 in the decarboxylation reaction (17). In this report, we describe the purification and characterization of a ferulic acid decarboxylase from P. fluorescens.
MATERIALS AND METHODSMaterials and reagents. trans-Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid, 99%), trans-cinnamic acid, hydrocinnamic acid, cis-2-methoxycinnamic acid, 2-carboxycinnamic acid, iodoacetamide, p-chloromercuribenzoic acid, N-ethylmaleimide, and diethyl pyrocarbonate were purchased from Aldrich Chemical Company (Milwaukee, Wis. Microorganism and culture conditions. The P. ...