title: Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.
abstract: A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.
authors.affiliation: Department of Microbiology, Michigan State University, East Lansing 48824, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Chee-Sanford
authors.forename: J W
authors.initials: JW
authors.lastname: Frost
authors.forename: M R
authors.initials: MR
authors.lastname: Fries
authors.forename: J
authors.initials: J
authors.lastname: Zhou
authors.forename: J M
authors.initials: JM
authors.lastname: Tiedje
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism*; D001673:Biodegradation, Environmental; D006092:Gram-Negative Facultatively Anaerobic Rods / Q000201:enzymology*; D009586:Nitrogen Fixation; D014050:Toluene / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D014050:Toluene; D000105:Acetyl Coenzyme A; C022903:cinnamoyl-coenzyme A