Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum.
A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.
He
Z
Z
Department of Microbiology, University of Georgia, Athens 30602-2605, USA.
Wiegel
J
J
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D002396:Catechols; D002262:Carboxy-Lyases; C099799:3,4-dihydroxybenzoate decarboxylase; C034221:catechol; D010100:Oxygen