Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.
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 (+/- 6%) 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 degrees 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.
Huang
Z
Z
Division of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City 52242.
Dostal
L
L
Rosazza
J P
JP
D000595:Amino Acid Sequence; D002021:Buffers; D002262:Carboxy-Lyases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism*; D006139:Guaiacol / Q000031:analogs & derivatives* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D002021:Buffers; D003373:Coumaric Acids; D008670:Metals; D006139:Guaiacol; C014245:4-vinylguaiacol; C004999:ferulic acid; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase