Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools

Abstract

We studied the benzylsuccinate synthase (Bss) reaction mechanism with respect to the hydrogen-carbon bond cleavage at the methyl group of toluene by using different stable isotope tools. ⌳ values (slopes of linear regression curves for carbon and hydrogen discrimination) for two-dimensional compound-specific stable isotope analysis (2D-CSIA) of toluene activation by Bsscontaining cell extracts (in vitro studies) were found to be similar to previously reported data from analogous experiments with whole cells (in vivo studies), proving that ⌳ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller ⌳ values than those of obligate anaerobes. In addition, a partial exchange of a single deuterium atom in benzylsuccinate with hydrogen was observed in experiments with deuterium-labeled toluene. In this study, the Bss enzymes of the tested facultative anaerobes showed 3-to 8-fold higher exchange probabilities than those for the enzymes of the tested obligate anaerobic bacteria. The phylogeny of the Bss variants, determined by sequence analyses of BssA, the gene product corresponding to the ␣ subunit of Bss, correlated with the observed differences in ⌳ values and hydrogen exchange probabilities. In conclusion, our results suggest subtle differences in the reaction mechanisms of Bss isoenzymes of facultative and obligate anaerobes and show that the putative isoenzymes can be differentiated by 2D-CSIA.

Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB

“…TS-6, “ A. aromaticum ” EbN1 and T. aromatica K172, and most probably they come from a microorganism, such as a Hermimiimonas -related strain, with a GC content lower than that of Thauera or Azoarcus strains. Interestingly, this subgrouping of Bss orthologs has also a reflect in the subtle differences observed in their reaction mechanism when using stable isotope tools ( Kummel et al, 2013 ).…”

“…Since alkylbenzenes are relatively soluble and mobile in water, they are widespread and tend to accumulate in hostile environments, e.g., subsurface anoxic environments, where they are of major concern to groundwater quality and ecosystem health. Therefore, the anaerobic degradation of alkylbenzenes is an important practical aspect of bioremediation and becomes crucial for the biogeochemical cycles ( Kummel et al, 2013 ; Rabus et al, 2016 ; Lueders, 2017 ). Despite the aerobic degradation of alkylbenzenes has been extensively studied, the microbial catabolism of these aromatic hydrocarbons under anaerobic conditions is much less known, and novel pathways and enzymes have been recently described, some of which are of potential biotechnological interest ( Rabus et al, 2016 ; Lueders, 2017 ).…”

Functional Gene Markers for Fumarate-Adding and Dearomatizing Key Enzymes in Anaerobic Aromatic Hydrocarbon Degradation in Terrestrial Environments

“…To develop concepts of population-based site management, a combination with other complementary strategies to assess biodegradation may become necessary, such as metabolite detection [Callaghan, 2013], the application of isotopic pathway identification [Kümmel et al, 2013] and of course high-throughput metagenomics and transcriptomics. Although the application of the latter has not been reported to date for an anoxic, aromatic compoundcontaminated aquifer, respective reports for other hydrocarbon-impacted systems are available Kimes et al, 2013;Luo et al, 2014;Tan et al, 2015].…”

Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase

“…Very similar initiation reactions by fumarate additions catalyzed by closely related glycyl radical enzymes have since been described for anaerobic degradation of xylenes, ethylbenzene, p -cresol, 2-methylnaphthalene, p -cymene, and even alkanes, which seem to be added to fumarate at their subterminal methylene groups (reviewed in [ 19 ]). BSS and the growing number of additional fumarate-adding enzymes have become model cases for environmental processes in contaminated soils and deep anoxic subsediment habitats in recent years, and their isotopic preferences and conserved sequences serving as templates for molecular probes are currently employed as tools for monitoring these processes in situ [ 20 , 21 , 22 , 23 ].…”