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.

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow

“…Both the pps and ppc cassettes share high sequence similarities (> 40%) and gene organizations with T. aromatica (Schmeling et al, 2004;Schuhle and Fuchs, 2004) (Supporting Information Table S3). These observations suggest that strain UI invests scarce ATP into phenol activation rather than performing ATP-independent phenol degradation as proposed for Sedimentibacter hydroxybenzoicus strain JW/Z-1 (Zhang and Wiegel, 1994;He and Wiegel, 1995) (Fig. 2).…”

Anaerobic Metabolism of Aromatic Compounds

“…These steps may complement the action of ATP hydrolysis, which is thought to lower dramatically the redox potential of the reduced-electron transferring group. Single-turnover studies 97 and studies with 33 Sand 57 Fe-labeled enzymes suggested the presence of a sulfur-centered species, most probably a disulfide radical anion formed by two cysteine residues in close proximity of an active-site [4Fe-4S] cluster.…”

Unusual reactions involved in anaerobic metabolism of phenolic compounds

“…Some of these proteins might be involved in the metabolism of similar phenolic compounds. The 54-kDa 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum (He and Wiegel, 1995;Huang et al, 1999) and subunits of various proven or putative vanillic acid (3-methoxy-4-hydroxybenzoic acid) decarboxylases from E. coli (Blattner et al, 1997), Bacillus subtilis (Cavin et al, 1998), and Streptomyces sp. (Chow et al, 1999) show similarity with UbiD and UbiX.…”