Benzoate-coenzyme A ligase, encoded by badA, is one of three 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 microM 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 microM 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) with 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.
Egland
P G
PG
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Gibson
J
J
Harwood
C S
CS
D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism*; D019817:Benzoic Acid; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D012241:Rhodopseudomonas / Q000201:enzymology* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; D001565:Benzoates; D003509:Cyclohexanecarboxylic Acids; C037817:benzoyl-coenzyme A; D019817:Benzoic Acid; C058481:4-hydroxybenzoate coenzyme A ligase; D003066:Coenzyme A Ligases; C055455:benzoate coenzyme A ligase; C044895:cyclohexanecarboxylic acid