title: Purification and characterization of an oxygen-sensitive reversible 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum.
abstract: A 4-hydroxybenzoate decarboxylase from the anaerobe Clostridium hydroxybenzoicum strain JW/Z-1T was purified and partially characterized. It had an apparent molecular mass of 350 kDa and consisted of six identical subunits of 57 kDa each. The temperature optimum for the decarboxylation was approximately 50 degrees C, the optimum pH 5.6-6.2. The pI of the enzyme was 5.1. The activation energy for decarboxylation of 4-hydroxybenzoate was 65 kJ.mol-1 (20-37 degrees C). The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate. The apparent Km and kcat values obtained for 4-hydroxybenzoate were 0.40 mM and 3.3 x 10(3) min-1, and for 3,4-dihydroxybenzoate 1.2 mM and 1.1 x 10(3) min-1, respectively, at pH 6.0 and 25 degrees C. The enzyme activity was not influenced by the addition of biotin or avidin to either the crude cell extracts or the purified enzyme. The p-hydroxyl group of hydroxybenzoate appears to be essential for binding by the enzyme. The N-terminal amino acid sequence shows some similarity to the uroporphyrinogen decarboxylases from Synechococcus and Saccharomyces. The enzyme catalyzed the reverse reactions, that is, the carboxylation of phenol to 4-hydroxybenzoate and of catechol to 3,4-dihydroxybenzoate. The carboxylation did not require ATP.
authors.affiliation: Department of Microbiology, University of Georgia, Athens 30602-2605, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: He
authors.forename: J
authors.initials: J
authors.lastname: Wiegel
mesh_terms: D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D002262:Carboxy-Lyases; C065828:4-hydroxybenzoate decarboxylase; D010100:Oxygen