Molecular cloning of 3-phenylcatechol dioxygenase involved in the catabolic pathway of chlorinated biphenyl from Pseudomonas putida and its expression in Escherichia coli

Appl Environ Microbiol. 1988 Nov;54(11):2664-71. doi: 10.1128/aem.54.11.2664-2671.1988.

Abstract

Genes encoding 3-phenylcatechol dioxygenases were cloned from the chlorobiphenyl-degrading Pseudomonas putida strain OU83, using broad-host-range cosmid vector pCP13. Restriction enzyme analysis of DNA from 2,3-dioxygenase-positive chimeric cosmids showed DNA inserts ranging in size from 6.0 to 30 kilobases. The origin of the DNA insert in hybrid clones was established by using 32P-labeled hybrid clones (pOH101 and pOH810). A 2.3-kilobase HindIII fragment was common to two clones. The 2,3-dioxygenase from the parent P. putida strain, OU83, and the recombinant clones (pOH101 and pOH8101) showed similar characteristics as determined by isoelectric focusing and polyacrylamide gel electrophoresis. The 2,3-dioxygenase from the Escherichia coli recombinant cosmid showed a pI of 5.0, a Km of 14 microM, and broad substrate activity with catechol, 4-chlorocatechol, 4-methylcatechol, and 2,3-dihydroxybiphenyl.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Biodegradation, Environmental
  • Biphenyl Compounds / metabolism*
  • Catechol 2,3-Dioxygenase
  • Catechols / metabolism
  • Cloning, Molecular
  • Cosmids
  • DNA, Bacterial / genetics
  • Dioxygenases*
  • Escherichia coli / genetics*
  • Gene Expression Regulation*
  • Genes, Bacterial
  • Nucleic Acid Hybridization
  • Oxygenases / genetics*
  • Pseudomonas / enzymology
  • Pseudomonas / genetics*
  • Pseudomonas / growth & development
  • Restriction Mapping
  • Sequence Homology, Nucleic Acid
  • Substrate Specificity

Substances

  • Biphenyl Compounds
  • Catechols
  • DNA, Bacterial
  • Oxygenases
  • 3-phenylcatechol dioxygenase
  • Dioxygenases
  • Catechol 2,3-Dioxygenase