Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47.

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.