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Journal of Bacteriology, January 2002, p. 207-215, Vol. 184, No. 1
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.1.207-215.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Stefan R. Kaschabek,1,{dagger} Bernd Kuhn,1 Dagmar Müller,2 Eberhard Schmidt,1 and Walter Reineke1*

Chemische Mikrobiologie, Bergische Universität-Gesamthochschule Wuppertal, Wuppertal,1 Außenstelle Jülich, Institut für Enzymtechnologie der Universität Düsseldorf, Düsseldorf Germany2

Received 16 July 2001/ Accepted 2 October 2001

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.

* Corresponding author. Mailing address: Bergische Universität-Gesamthochschule Wuppertal, Chemische Mikrobiologie, Fachbereich 9, Gaußstraße 20, D-42097 Wuppertal, Germany. Phone: 49-202-4392456. Fax: 49-202-4392698. E-mail: reineke{at}uni-wuppertal.de.

{dagger} Present address: Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, D-09599 Freiberg, Germany.

Journal of Bacteriology, January 2002, p. 207-215, Vol. 184, No. 1
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.1.207-215.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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