The 6.8-kb XhoI fragment of chromosomal DNA of Pseudomonas sp. DJ77 contains the phnDEFG genes involved in the degradation of polyaromatic hydrocarbons and chlorinated aromatics. Here, we report the nucleotide sequence of the phnD gene encoding a 2-hydroxymuconic semialdehyde hydrolase and its substrate specificity. The PhnD hydrolase contains 286 amino acids with a M(r) of 31301. The deduced amino acid sequence of the PhnD enzyme is 31.0-50.5% identical to those of homologous enzymes encoded by the dmp, tod, xyl, and bph operons. The PhnD enzyme is required for conversion of 2-hydroxymuconic semialdehyde, which is produced from catechol by the PhnE catechol 2,3-dioxygenase, to 2-hydroxypent-2,4-dienoate. We now confirm that the phnD gene is located immediately upstream of the catechol 2,3-dioxygenase gene (phnE) unlike other meta-cleavage operons.