Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter.
We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-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 gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-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 gamma-RA and carboxylation of RE.
Ishii
Yoshitaka
Y
Department of Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Narimatsu
Yoshiki
Y
Iwasaki
Yuichiro
Y
Arai
Naoki
N
Kino
Kuniki
K
Kirimura
Kohtaro
K
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D001234:Aspergillus niger / Q000378:metabolism; D001665:Binding Sites; D019735:Carbon-Carbon Ligases / Q000737:chemistry; D002262:Carboxy-Lyases / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D002396:Catechols / Q000737:chemistry; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D012118:Resorcinols / Q000737:chemistry*; D020578:Sphingomonas / Q000378:metabolism; D013696:Temperature; D014212:Tretinoin / Q000737:chemistry
D002264:Carboxylic Acids; D002396:Catechols; D062385:Hydroxybenzoates; D012118:Resorcinols; D006639:Histidine; D014212:Tretinoin; D004247:DNA; C466306:5-carboxyvanillate decarboxylase; D002262:Carboxy-Lyases; C493926:gamma-resorcylic acid decarboxylase, Rhizobium radiobacter; D019735:Carbon-Carbon Ligases; D003545:Cysteine; C034221:catechol; C412605:gamma-resorcylic acid; D010100:Oxygen