BENZOATE-COENZYME A LIGASE, ENCODED BY BADA, IS ONE OF 3 LIGASES ABLE TO CATALYZE BENZOYL-COENZYME A FORMATION DURING ANAEROBIC GROWTH OF RHODOPSEUDOMONAS-PALUSTRIS ON BENZOATE

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 mu M benzoate, To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced, The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R, palustris. A badA mutant that was constructed had barely detectable levels of Ligase activity when cell extracts were assayed at 10 mu M benzoate, Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s),vith a relatively low affinity for benzoate, A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant, Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme, These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate, This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.