In the denitrifying member of the ␤-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acylCoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoateCoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V max /K m, the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate.

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.