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Degradation of Aromatics and Chloroaromatics by Pseudomonas sp . Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase. Markus Göbel, 2002.3-Oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway . This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp . strain B13 that encode these enzymes . DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one . ORF1, of unknown function, has a length of 414 bp . ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813 . ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661 . CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a . ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity . The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF . Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF . ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase . An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found . The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature .
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