Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31

Abstract

Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930-5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2, 3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.

FIG. 1

A schematic representation of the organization of the ORFs that were identified on the 3.1-kb PstI fragment. The PstI cloning sites and a XhoI site that was confirmed by digestion are shown. The positions of the start codons that were identified are indicated, as well as the sizes of the three complete ORFs. The position where a deletion of monooxygenase genes that corresponded to tbmBCDE (17) may have occurred is indicated with an arrow.

FIG. 2

Structure-based amino acid sequence alignment of CbzE with those of other extradiol dioxygenases. The secondary structure elements are indicated below the sequence of BphC as open bars (for α helices) and arrows (for β strands). The alignment was based on the structure-validated alignment that was made by Eltis and Bolin (9). Residues that are strictly conserved within the catechol 2,3-dioxygenases are shown in boldface type, and the three amino acids that are known to be involved in the binding of Fe²⁺ are underlined. Sequences (GenBank accession numbers in parentheses): CbzE, chlorocatechol 2,3-dioxygenase of P. putida GJ31; TdnC, 3-methylcatechol 2,3-dioxygenase of P. putida UCC2 (X59790) (unpublished data); C23O, Catechol 2,3-dioxygenase of B. cepacia AA1 (U47111) (unpublished data); TomB, catechol 2,3-dioxygenase of B. cepacia G4 (36); Cdo2, catechol 2,3-dioxygenase II of P. putida MT15 (U01286) (unpublished data); TbuE, catechol 2,3-dioxygenase of R. pickettii PKO1 (U20258) (23); XylE, catechol 2,3-dioxygenase of P. putida mt-2 (V01161) (31); BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase of B. cepacia LB400 (X66122) (14).

FIG. 3

Specific production of 2-hydroxymuconate (2HM) by several catechol 2,3-dioxygenases (C23Os) due to the conversion of 3CC (100 μM). The production of 2HM was measured spectrophotometrically at 290 nm. The amount of C23O that was added was determined as the amount of C23O activity with catechol. Lines: ———, CbzE; · ⋯, TdnC, —⋯—, C23OII; — —, XylE.

FIG. 4

Formation of meta-cleavage products due to conversion of catechol (———), 3MC (⋯), and 4MC (— —) by CbzE (A), TdnC (B), and XylE (C). The production of the meta-cleavage compounds was measured spectrophotometrically at 375, 388, and 382 nm for catechol, 3MC, and 4MC, respectively. For CbzE, the product formation was linear in time with each catechol. For TdnC and XylE, the rates of product formation with 3MC and 4MC decreased over time due to inactivation of the enzyme.

FIG. 5

Conversion of 3CC by CbzE and XylE. The open circles and squares show the concentrations of 3CC that were measured in the assay mixtures with CbzE and XylE, respectively. The lines show the 3CC depletion curves that were fitted through the data with equation 1.

FIG. 6

Schematic representation of the construction of hybrid catechol 2,3-dioxygenases (H1 to H10). The primers (P1 to P14) that were used in PCRs to generate DNA fragments that contained overlapping regions for the PCR fusions are indicated with arrows. □, polypeptide fragments derived from CbzE; , polypeptide fragments derived from TdnC. The amino acids of CbzE and TdnC between which the fusions were made are indicated.