Reversible and nonoxidative γ-resorcylic acid decarboxylase: Characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

We found a γ-resorcylic acid (γ-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form γ-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of γ-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of γ-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with γ-RA. The enzyme, γ-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of γ-RA, but not α-RA or β-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form γ-RA, without formation of α-RA and β-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this γ-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of γ-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the γ-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of γ-RA and carboxylation of RE.