A novel 2,4-dichlorophenol hydroxylase (TfdB, EC 1.14.13.20) gene, designated as tfdB-JLU, was identified from a metagenome constructed from polychlorinated biphenyl-contaminated soil by functional screening and heterologously expressed in Escherichia coli. The deduced amino acid sequence of tfdB-JLU exhibited less than 48% homology with other known TfdBs. The enzyme exhibited a wider substrate spectrum than the previously reported TfdBs and higher relative activity towards ortho-substituted dichlorophenols, 2-chlorophenol, and 3-chlorophenol than towards 2,4-dichlorophenol, the preferred substrate of other known TfdBs. The enzyme had a K-m of 5 mu M for 2,4-dichlorophenol and 6 mu M for NADPH. The optimal temperature and pH of the enzyme were 25 degrees C and 7.5, respectively. Activity of the purified TfdB-JLU was slightly enhanced by Ca2+, Mn2+, Co2+, and Fe2+, and completely inhibited by Cu2+, Hg2+, and Zn2+. This study is the first report to identify a novel TfdB from a metagenome.