Characterization of the pcbD Gene Encoding 2-Hydroxy-6-Ox0-6-Phenylgexa-2,4-Dienoate Hydrolase from Pseudomonas sp. P20

2-Hydroxy-6-oxo-6phenylhexa-2,4-dienoate (HOPDA) hydrolase catalyzes the hydrolytic cleavage of HOPDA to bemzpate and 2-hydroxypenta-2, 4-dienoate (HPD) during microbial catabolism of biphenyl and polychlorinated biphenyls. A HOPDA hydrolase gene (pcbD) was isolated from the genomic library of Pseudomonas sp. P20 and designated as pCNUO1201; a 7.5-kb XbaI DNA fragment from Pseudomonas sp. P20 was inserted into the pBluescript SK(+) XbaI site. E. coli HB101 harboring pCNU1201 exhibited HOPDA hydrolase activity. The open reading frame (ORF) corresponding to the pcbD gene consisted of 855 base pairs with an ATG initiation codon and a TGA termination codon. The ORF was preceded by a rebosome-binding sequence of 5'-TGGAGC-3' and its G+C content was 55 mol%. The pcbD gene of Pseudomonas sp. P20 was located immedeately downstream of the pcbC gene encoding 2,3- dihydroxybiphenyl 1,2-dioxygenase, and approximately 4-kb upstream of the pcbE gene encoding HPD hydratase. The pcbK gene was able to encode a polypeptide with a molecular weight of 31,732 containing 284 amino acid residues. The deduced amino acid sequence of the HOPDA hydrolase of Pseudomonas sp. P20 exhibited high identity (62%) with those of the HOPDA hydrolases of P. putida KF715, P. pseudoalcaligenes KF707, and Burkholderia cepacia LB400, and also significant homology with those of other hydrolytic enzymes including esterase, transferase, and peptidase.