title: Involvement of coenzyme A thioesters in anaerobic metabolism of 4-hydroxybenzoate by Rhodopseudomonas palustris.
abstract: The initial steps of anaerobic 4-hydroxybenzoate degradation were studied in whole cells and cell extracts of the photosynthetic bacterium Rhodopseudomonas palustris. Illuminated suspensions of cells that had been grown anaerobically on 4-hydroxybenzoate and were assayed under anaerobic conditions took up [U-14C]4-hydroxybenzoate at a rate of 0.6 nmol min-1 mg of protein-1. Uptake occurred with high affinity (apparent Km = 0.3 microM), was energy dependent, and was insensitive to external pH in the range of 6.5 to 8.2 Very little free 4-hydroxybenzoate was found associated with cells, but a range of intracellular products was formed after 20-s incubations of whole cells with labeled substrate. When anaerobic pulse-chase experiments were carried out with cells incubated on ice or in darkness, 4-hydroxybenzoyl coenzyme A (4-hydroxybenzoyl-CoA) was formed early and disappeared immediately after addition of excess unlabeled substrate, as would be expected of an early intermediate in 4-hydroxybenzoate metabolism. A 4-hydroxybenzoate-CoA ligase activity with an average specific activity of 0.7 nmol min-1 mg of protein-1 was measured in the soluble protein fraction of cells grown anaerobically on 4-hydroxybenzoate. 4-Hydroxybenzoyl-CoA was the sole product formed from labeled 4-hydroxybenzoate in the ligase reaction mixture. 4-Hydroxybenzoate uptake and ligase activities were present in cells grown anaerobically with benzoate, 4-hydroxybenzoate, and 4-aminobenzoate and were not detected in succinate-grown cells. These results indicate that the high-affinity uptake of 4-hydroxybenzoate by R. palustris is due to rapid conversion of the free acid to its CoA derivative by a CoA ligase and that this is also the initial step of anaerobic 4-hydroxybenzoate degradation.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, New York 14853.
authors.forename: S M
authors.initials: SM
authors.lastname: Merkel
authors.forename: A E
authors.initials: AE
authors.lastname: Eberhard
authors.forename: J
authors.initials: J
authors.lastname: Gibson
authors.forename: C S
authors.initials: CS
authors.lastname: Harwood
mesh_terms: D000214:Acyl Coenzyme A / Q000138:chemical synthesis; D000332:Aerobiosis; D000693:Anaerobiosis; D002250:Carbon Radioisotopes; D003066:Coenzyme A Ligases / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism*; D007700:Kinetics; D010226:Parabens; D012241:Rhodopseudomonas / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D062385:Hydroxybenzoates; D010226:Parabens; C058482:4-hydroxybenzoyl-coenzyme A; C058481:4-hydroxybenzoate coenzyme A ligase; D003066:Coenzyme A Ligases; C038193:4-hydroxybenzoic acid