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

The first step of anaerobic benzoate degradation is the formation of b enzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 mu M benz oate, To study the molecular basis for this reaction, the benzoate-coe nzyme 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-hydr oxybenzoate-coenzyme A ligase (50% amino acid identity) from R, palust ris. A badA mutant that was constructed had barely detectable levels o f Ligase activity when cell extracts were assayed at 10 mu M benzoate, Despite this, the mutant grew at wild-type rates on benzoate under la boratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentratio n of benzoate (1 mM). This suggested that R. palustris expresses, in a ddition 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 constr ucting a badA hbaA double mutant, Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significa nt, suggesting the involvement of a third enzyme in benzoate activatio n. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexan ecarboxylate-coenzyme A ligase as being this third enzyme, These resul ts show that wild-type R. palustris cells synthesize at least three en zymes that can catalyze the initial step in anaerobic benzoate degrada tion during growth on benzoate, This observation supports previous sug gestions that benzoyl-coenzyme A formation plays a central role in ana erobic aromatic compound biodegradation.