Molecular cloning of 3-phenylcatechol dioxygenase involved in the catabolic pathway of chlorinated biphenyl from Pseudomonas putida and its expression in Escherichia coli.
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.
Khan
A
A
Department of Biological Sciences, Oakland University, Rochester, Michigan 48309-4401.
Tewari
R
R
Walia
S
S
D001673:Biodegradation, Environmental; D001713:Biphenyl Compounds / Q000378:metabolism*; D050557:Catechol 2,3-Dioxygenase; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000235:genetics; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009693:Nucleic Acid Hybridization; D010105:Oxygenases / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D001713:Biphenyl Compounds; D002396:Catechols; D004269:DNA, Bacterial; D010105:Oxygenases; C058196:3-phenylcatechol dioxygenase; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase