Characterization of the 4-hydroxybenzoyl-coenzyme A thioesterase from Arthrobacter sp. strain SU

Abstract

The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k(cat) of 6.7 s(-1) and a K(m) of 1.2 micro M. The k(cat) pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.

FIG. 1.

Illustration of the reaction steps of the bacterial 4-CBA dehalogenation pathway (28).

FIG. 2.

Coomassie blue-stained SDS-PAGE gel of purified Arthrobacter sp. strain SU 4-HBA-CoA thioesterase. Lane 1 shows the Invitrogen benchmark protein ladder. Lane 2 shows the chromatography of the purified Arthrobacter sp. strain SU 4-HBA-CoA thioesterase.

FIG. 3.

pH profile of the k_cat for the 4-HBA-CoA thioesterase from Arthrobacter sp. strain SU. See Materials and Methods for details.

FIG. 4.

Twenty representative sequences derived from the GCG PILE-UP of the sequence homologues to the Arthrobacter sp. strain SU 4-HBA-CoA thioesterase (Arthio) at the top and the Pseudomonas sp. strain CBS3 4-HBA-CoA thioesterase (Psthio) at the bottom. The protein sequences are labeled with their accession numbers (NCBI protein database).