Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product

Abstract

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

FIG. 1

Proposed reactions involved in anaerobic toluene oxidation to benzoyl-CoA. This pathway is based on research conducted with denitrifying strain T (2) and T. aromatica (5) and sulfate-reducing strain PRTOL1 (3).

FIG. 2

Chiral HPLC analysis of benzylsuccinate formed in vitro from toluene and fumarate. (A) Sample containing benzylsuccinate produced from toluene and fumarate by permeabilized strain T cells (see text for experimental conditions); (B) the sample coinjected with a (+)-benzylsuccinate standard (0.8 mM); (C) the sample coinjected with a (−)-benzylsuccinate standard (0.8 mM). The identity of the compound eluting at ∼21 min is unknown.

FIG. 3

Proposed reaction mechanism for benzylsuccinate synthase. E represents the enzyme. Note that the exact same H atom is bound to the enzyme in II and III.