The glycyl radical enzyme-catalyzed addition of <i>n</i>-alkanes to fumarate creates a C-C-bond between two concomitantly formed stereogenic carbon centers. The configurations of the two diastereoisomers of the product resulting from <i>n</i>-hexane activation by the <i>n</i>-alkane-utilizing denitrifying bacterium strain HxN1, i.e. (1-methylpentyl)succinate, were assigned as (2<i>S</i>,1′<i>R</i>) and (2<i>R</i>,1′<i>R</i>). Experiments with stereospecifically deuterated <i>n</i>-(2,5-<sup>2</sup>H<sub>2</sub>)hexanes revealed that exclusively the pro-<i>S</i> hydrogen atom is abstracted from C2 of the <i>n</i>-alkane by the enzyme and later transferred back to C3 of the alkylsuccinate formed. These results indicate that the alkylsuccinate-forming reaction proceeds with an inversion of configuration at the carbon atom (C2) of the <i>n</i>-alkane forming the new C-C-bond, and thus stereochemically resembles a S<sub>N</sub>2-type reaction. Therefore, the reaction may occur in a concerted manner, which may avoid the highly energetic hex-2-yl radical as an intermediate. The reaction is associated with a significant primary kinetic isotope effect (kH/kD ≥3) for hydrogen, indicating that the homolytic C-H-bond cleavage is involved in the first irreversible step of the reaction mechanism. The (1-methylalkyl)succinate synthases of <i>n</i>-alkane-utilizing anaerobic bacteria apparently have very broad substrate ranges enabling them to activate not only aliphatic but also alkyl-aromatic hydrocarbons. Thus, two denitrifiers and one sulfate reducer were shown to convert the nongrowth substrate toluene to benzylsuccinate and further to the dead-end product benzoyl-CoA. For this purpose, however, the modified β-oxidation pathway known from alkylbenzene-utilizing bacteria was not employed, but rather the pathway used for <i>n</i>-alkane degradation involving CoA ligation, carbon skeleton rearrangement and decarboxylation. Furthermore, various <i>n</i>-alkane- and alkylbenzene-utilizing denitrifiers and sulfate reducers were found to be capable of forming benzyl alcohols from diverse alkylbenzenes, putatively via dehydrogenases. The thermophilic sulfate reducer strain TD3 forms <i>n</i>-alkylsuccinates during growth with <i>n</i>-alkanes or crude oil, which, based on the observed patterns of homologs, do not derive from a terminal activation of <i>n</i>-alkanes.
Molecular Basis of C–S Bond Cleavage in the Glycyl Radical Enzyme Isethionate Sulfite-Lyase
