ANAEROBIC ACTIVATION OF TOLUENE AND O-XYLENE BY ADDITION TO FUMARATE IN DENITRIFYING STRAIN-T

Anaerobic assays conducted with strain T, a denitrifying bacterium cap able of mineralizing toluene to carbon dioxide, demonstrated that tolu ene-grown, permeabilized cells catalyzed the addition of toluene to fu marate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP, In the presence of CoA, formatio n of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate format ion from toluene and fumarate in assays with permeabilized cells was > 30% of the specific rate of toluene consumption in whole-cell suspensi ons with nitrate; this observation suggests that benzylsuccinate forma tion may be the first reaction in anaerobic toluene degradation by str ain T. Use of deuterium-labeled toluene and gas chromatography-mass sp ectrometry indicated that the H atom abstracted from the toluene methy l group during addition to fumarate was retained in the succinyl moiet y of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-C oA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl) succinate. o -Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rat es, the observed characteristics of the toluene-fumarate addition reac tion (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction, Thus, add ition to fumarate may be a biochemical strategy to anaerobically activ ate a range of methylbenzenes.