Substrate range of benzylsuccinate synthase from Azoarcus sp strain T

Benzylsuccinate synthase, which catalyzes the anaerobic addition of the met hyl carbon of toluene to fumarate, has recently been reported in several de nitrifying and sulfate-reducing, toluene-degrading bacteria. In substrate r ange studies with partially purified benzylsuccinate synthase from denitrif ying Azoarcus sp, strain T, benzylsuccinate analogs were observed as a resu lt of fumarate addition to the following toluene surrogates: xylenes, monof luorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-1 -cyclohexene or methylcyclohexane). Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from a ssays with toluene and either crotonate or trans-glutaconate. Toluene-malea te addition, like toluene-fumarate addition, resulted in highly stereospeci fic formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-car boxypropionic acid]. The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccin ate was found to apply to several toluene surrogates. The implications of t hese observations for the mechanism of benzylsuccinate synthase will be dis cussed. (C) 1999 Federation of European Microbiological Societies. Publishe d by Elsevier Science B.V. All rights reserved.