Purification and characterization of 2,4-dichlorophenol hydroxylase isolated from a bacterium of the α-2 subgroup of the Proteobacteria

Abstract

2,4-Dichlorophenol hydroxylase (EC 1.14.13.20) was purified to apparent homogeneity from the bacterial strain S1, a member of the alpha-2 subgroup of the Proteobacteria. The molecular masses of the native enzyme and the subunit were determined to be 256 and 64 kDa, respectively, suggesting a homotetrameric structure. The enzyme converted 2,4-dichlorophenol to 3,5-dichlorocatechol. The apparent K(m) values for 2,4-dichlorophenol, NADPH and NADH were 3, 240 and 420 microM, respectively. The enzyme hydroxylated a broad range of halogenated phenols. 3-Chloro-, 2,6-dichloro- and 2,4,6-trichlorophenol acted as 'non-substrate' effectors. The N-terminal sequence revealed 72% identity with the amino acid sequence deduced from the pJP4-encoded tfdB gene.

Cloning and characterisation of a novel 2,4-dichlorophenol hydroxylase from a metagenomic library derived from polychlorinated biphenyl-contaminated soil

“…The enzyme also showed higher activity towards 2,5-dichlorophenol, 2,3-dichlorophenol, 2,6-dichlorophenol, and 2-chlorophenol than towards 2,4-DCP, which is the preferred substrate of all previously reported TfdBs. These features are not found in previously characterised TfdBs (Beadle and Smith 1982;Makdessi and Lechner 1997;Ledger et al 2006;Huong et al 2007), which exhibit high oxidation activities towards 2,4-DCP and low relative activities towards other chlorophenols. Thus, TfdB-JLU is a promising potential enzyme for bioremediation of chlorophenol-contaminated environments.…”

“…The identified TfdB, designated as TfdB-JLU, shared less than 48% amino Beadle and Smith (1982) e Makdessi and Lechner (1997) f Huong et al (2007) acid sequence identity with other known TfdBs. In addition, the purified TfdB-JLU exhibited a wider substrate spectrum than other TfdBs and higher relative activity towards the ortho-substituted dichlorophenols, 2-chlorophenol, and 3-chlorophenol than towards 2,4-DCP, the preferred substrate of other TfdBs.…”

“…The K m for NADPH (6 lM) was much lower than that for NADH (330 lM), but the V max for NADPH (0.039 lM min -1 ) was only 1.9-fold higher than that for NADH (0.021 lM min -1 ). Since the affinity (1/K m ) and catalytic efficiency (V max /K m ) for 2,4-DCP in NADPH were higher than in NADH at the same concentration, the purified enzyme appeared to prefer NADPH over NADH as an external electron donor, similar to other TfdBs (Liu and Chapman 1984;Makdessi and Lechner 1997).…”

“…The values for NADH and NADPH were determined using 2,4-DCP at 0.1 mM with NADH or NADPH from 5 to 2,000 lM (Makdessi and Lechner 1997). The kinetic constants were calculated from Lineweaver-Burk plots via non-linear regression using GraphPad Prism 5 (GraphPad, San Diego, CA).…”

“…Although many chlorophenol hydroxylases have been isolated from different genera, only a few TfdB enzymes have been purified and characterised (Beadle and Smith 1982;Liu and Chapman 1984;Radjendirane et al 1991;Makdessi and Lechner 1997;Ledger et al 2006;Huong et al 2007). Their substrate specificities are similar, and most of them display high activity toward 2,4-DCP and 4-chloro-2-methylphenol.…”