Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31.
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.
Mars
A E
AE
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
Kingma
J
J
Kaschabek
S R
SR
Reineke
W
W
Janssen
D B
DB
D000595:Amino Acid Sequence; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism*; D003001:Cloning, Molecular; D049308:Dioxygenases; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity