Degradation of aromatics and chloroaromatics by Pseudomonas sp. strain B13: purification and characterization of 3-oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase

J Bacteriol. 2002 Jan;184(1):207-15. doi: 10.1128/JB.184.1.207-215.2002.


The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 +/- 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 +/- 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl-CoA C-Acyltransferase / isolation & purification
  • Acetyl-CoA C-Acyltransferase / metabolism*
  • Adipates / metabolism*
  • Amino Acid Sequence
  • Benzoates / metabolism*
  • Biodegradation, Environmental
  • Chlorobenzenes / metabolism*
  • Coenzyme A-Transferases / isolation & purification
  • Coenzyme A-Transferases / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Pseudomonas / enzymology*
  • Sequence Analysis, Protein
  • Substrate Specificity


  • Adipates
  • Benzoates
  • Chlorobenzenes
  • Acetyl-CoA C-Acyltransferase
  • 3-oxoadipyl-coenzyme A thiolase
  • Coenzyme A-Transferases
  • 3-oxoadipate CoA-transferase