Molecular analysis of aerobic phenylacetate degradation in Azoarcus evansii

The Azoarcus evansii gene which codes for phenylacetate-CoA ligase, an enzyme involved in the aerobic degradation of phenylacetate, was isolated from a genomic library, using as the probe a fragment of the gene which encodes the isoenzyme that is induced under anaerobic conditions. By this means both the gene and its flanking sequences were recovered. The gene is homologous to the phenylacetate-CoA ligase genes of Pseudomonas putida U and Escherichia coli W. Induction by phenylacetate under aerobic growth conditions was demonstrated using lacZ fusions. Western analysis showed that phenylacetate-CoA ligase is involved in the degradation of the aromatic amino acid phenylalanine. Genes coding for the phenylacetate-CoA ligase and for the putative hydroxylating enzyme were expressed in E. coli. Detection of 2-hydroxyphenylacetate in the recombinant E. coli strain indicated hydroxylation of phenylacetyl-CoA. The gene pacL, which codes for the putative ring-opening enzyme was mutated to enable the isolation of intermediates in aerobic phenylacetic acid degradation, which were characterized by GC-MS and NMR analyses.