Thermophilic, Reversible γ-Resorcylate Decarboxylase from Rhizobium sp. Strain MTP-10005: Purification, Molecular Characterization, and Expression

Abstract

We found the occurrence of thermophilic reversible ␥-resorcylate decarboxylase (␥-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of ␥-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k cat /K m (m⌴ ؊1 ⅐ s ؊1 ) for ␥-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from

Metabolism of 2-hydroxy-1-naphthoic acid and naphthalene via gentisic acid by distinctly different sets of enzymes in Burkholderia sp. strain BC1

“…The enzymic transformation of 2H1NA to 2-naphthol is attributed to the action of a non-oxidative decarboxylase. Similar enzyme activity was noted for the decarboxylation of other aromatic acids (Gu et al, 2011;Matsui et al, 2006;Yoshida et al, 2004). Formation of 1-naphthol from 1H2NA has previously been documented in the metabolism of phenanthrene (Feng et al, 2012, and references therein), but this is the first report on the bacterial metabolism of 2H1NA via 2-naphthol.…”

“…Inhibition of enzyme activity by a histidine residue-specific inhibitor indicates that 2H1NA decarboxylase in strain BC1 possibly belongs to the metal-dependent hydrolase superfamily as reported in c-resorcylate decarboxylase (Ishii et al, 2004;Yoshida et al, 2004), 2,3-dihydroxybenzoate decarboxylase (Kamath et al, 1989) and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Li et al, 2006). Decarboxylases VII, catechol; VIII, salicylic acid; IX, 2H1NA; X, 1,2,6-trihydroxy-1,2-dihydronaphthalene; XI, 1,2,6-trihydroxynaphthalene; XII, 2-hydroxy-4-(29-oxo-5-hydroxy-3,5-cyclohexadienyl)-buta-2, 4-dienoate; XIII, cis-2,5-dihydroxybenzylidenepyruvate; XIV, maleyl pyruvic acid; XV, naphthalene; XVI, 1,2-dihydroxy-1,2-dihydronaphthalene; XVII, 1,2-dihydroxynaphthalene; XVIII, 2-hydroxy-4-(29-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoate; XIX, cis-ohydroxybenzylidenepyruvate; XX, cis,cis-muconic acid.…”

“…On: Sat, 12 May 2018 13:32:29 belonging to this superfamily are insensitive to oxygen and their subunit molecular masses are in the range 28-39 kDa (Ishii et al, 2004;Li et al, 2005;Yoshida et al, 2004). A 12.5 % SDS-PAGE analysis revealed a differentially overexpressed~32 kDa intense band in the crude enzyme preparations of 2H1NA-grown cells in comparison with that in the 2-naphthol-or succinate-grown preparations (Fig.…”

Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

“…This family includes ACMS decarboxylase from bacteria, animals, and humans (19,39,62); uracil-5-carboxylate decarboxylase of Neurospora crassa OR74A (57); the ␥-resorcylate decarboxylase GraF from Rhizobium sp. strain MTP-10005 (66,67); the 5-carboxyvanillate decarboxylase LigW (46); the biphenyl meta-cleavage compound hydrolase LigY (45); and the 4-oxalomesaconate hydratase LigJ (22) from Sphingobium sp. strain SYK-6.…”

C−H Carboxylation of Aromatic Compounds through CO2 Fixation

“…2,6‐Dihydroxybenzoate decarboxylase (2,6‐DHBD_Rs; Rhizobium sp . )54 and salicylic acid decarboxylase (SAD Tm; Trichosporon moniliiforme )55 were also found to be efficient for the carboxylation process. The phenolic acid decarboxylase derived from Lactobacillus plantarum (PAD_Lp)56 and Bacillus amyloliquefaciens (PAD_Ba)57 selectively acted at the β‐carbon atom of styrenes forming ( E )‐cinnamic acids.…”