Evaluation of Toluene Degradation Pathways by Two-Dimensional Stable Isotope Fractionation

Abstract

Toluene degradation by several pure and mixed microbial cultures was investigated bytwo-dimensional compound specific isotope analysis (2D-CSIA). For most of the cultures, the respective toluene degradation pathway and toluene attacking enzymatic step was known. The slope of the linear regression for hydrogen (delta delta(2)H) vs. carbon (delta delta(13)C) discrimination (lamda = delta delta(2)H/ delta delta(13)C approximately epsilonH(bulk)/epsilonC(bulk)) was determined in order to characterize aerobic and anaerobic toluene degradation pathways. The highest lamda value was estimated for the monohydroxylation of the methyl group by Pseudomonas putida (lamda = 53 +/- 5). The lowest value was observed for Rhodococcus opacus (lamda = 2 +/- 2) due to its insignificant hydrogen fractionation, which indicates that a ring dioxygenase was responsible for the initial attack of toluene. The fungus Cladosprium sphaerospermum containing a cytochrome P450-dependent methyl monooxygenase grouped within these extreme values (lamda = 16 +/- 6). Lamda values for organisms attacking toluene under anoxic conditions by benzylsuccinate synthase were significantly different and ranged from lamda = 4 +/- 3 (Blastochloris sulfoviridis) to 31 +/- 11 (strain TRM1). Values were in the same range for organisms using nitrate (lamda = 11-14) or sulfate (lamda = 28-31) as electron acceptor, indicating that it might be possible to distinguish toluene degradation under different electron acceptor conditions by 2D-CSIA.

Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H 2 and CO 2

“…3) A phylogeny based on functional genes rather than 16S rRNA gave a better correlation to fractionation factors of dichloromethane-degrading methylotrophic bacteria, as well as toluene-degrading microorganisms (46,47). However, the acetyl-CoA pathway has two distinct initial CO 2 -fixing enzymes (formate dehydrogenase and the carbon monoxide dehydrogenase/acetyl-CoA synthetase complex [48]), which both may contribute to the observed fractionation (Gelwicks et al [2] reported that both branches of the acetyl-CoA pathway show a similar fractionation).…”

Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools

“…S1D), the isotope fractionation patterns (characterized by the ⌳ values) are probably influenced by partial reactions preceding the C-H bond cleavage reaction, thus generating various AKIE values. Therefore, we propose that toluene transformation by Bss isoenzymes of obligate and facultative anaerobes may occur via slightly different reaction paths leading to the rate-limiting transition state preceding the C-H cleavage (23). The current mechanistic model for the Bss reaction mechanism predicts that the cleavage of the C-H bond is preceded by the formation of a thiol radical intermediate (see Fig.…”

“…Although toluene in the investigated cultures was always activated by benzylsuccinate synthases, the determined ⌳ values were significantly different, indicating that Bss subtypes in the investigated cultures may show slightly different reaction mechanisms. For facultative anaerobic toluene degraders, the observed ⌳ values were generally smaller than those for obligate anaerobic toluene degraders (23). This result motivated us to investigate the isotope-sensitive steps in the overall reaction in more detail.…”

“…Usually, isotope fractionation in complex biogeochemical systems is influenced by nonfractionating processes prior to the isotope-sensitive bond cleavage (e.g., transport or binding of the substrate to the enzyme), so the resulting apparent kinetic isotope effects (AKIEs) are often smaller than their corresponding KIEs (22). Accordingly, these processes may lead to a masking of the KIEs, frequently resulting in observed variabilities of AKIEs for specific enzymatic reactions (23,24). Masking effects can be canceled out by two-dimensional isotope fractionation analysis (2D-CSIA); in this case, the enrichments of the isotopic signatures of two elements in a residual substrate fraction during the reaction are compared and expressed by the lambda (⌳) value, which is defined as the slope of the linear regression for carbon and hydrogen discrimination (25).…”

Carbon and hydrogen isotope fractionation during nitrite-dependent anaerobic methane oxidation by Methylomirabilis oxyfera

“…Thereby, different pathways of substrate conversion can often be distinguished by the specific isotopic signature of the remaining pool or the product formed Mahieu et al, 2006;Meckenstock et al, 2004;Vogt et al, 2008).…”