Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d₈ and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d₈ and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = −1.219), Desulfobacterium cetonicum (b = −1.196), Thauera aromatica (b = −0.816), and Geobacter metallireducens (b = −1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = −2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the ¹³C/¹²C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d₈ and nonlabeled toluene for sulfate-reducing strain TRM1 (b = −0.728) and D. cetonicum (b = −0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37°C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20°C (b = −4.086) and minimum fractionation at 35°C (b = −2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d₅ in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)⁻¹. The D/H isotope fractionation (b = −1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.     

Carbon and hydrogen stable isotope fractionation during aerobic bacterial degradation of aromatic hydrocarbons

13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons

¹³C/¹²C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Stable isotope fractionation caused by glycyl radical enzymes during bacterial degradation of aromatic compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (epsilon) of -1.5 and -3.9, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic epsilon (epsilon(intrinsic)) were calculated. A comparison of epsilon(intrinsic) at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific epsilon elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of epsilon found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average epsilon if no fractionation factor is available for single compounds.     

Nitrogen removal by co-occurring methane oxidation,denitrification, aerobic ammonium oxidation,and anammox

Quantification of microbial contaminant biodegradation based on stable isotope fractionation analysis (SIFA) relies on known, invariable isotope fractionation factors. The microbially induced isotope fractionation is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. However, a number of non-isotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process and reduce the observed (i.e., the macroscopically detectable) isotope fractionation. This introduces uncertainty to the use of isotope fractionation for the quantification of microbial degradation processes. Here, we report on the influence of bacterial cell density on observed stable isotope fractionation. Batch biodegradation experiments were performed under non-growth conditions to quantify the toluene hydrogen isotope fractionation by exposing Pseudomonas putida mt-2(pWWO) at varying cell densities to different concentrations of toluene. Observed isotope fractionation depended significantly on the cell density. When the cell density rose from 5 × 10⁵ to 5 × 10⁸cells/mL, the observed isotope fractionation declined by 70% and went along with a 55% decrease of the degradation rates of individual cells. Theoretical estimates showed that uptake-driven diffusion to individual cells depended on cell density via the overlap of the cells’ diffusion-controlled boundary layers. Our data suggest that biomass effects on SIFA have to be considered even in well-mixed systems such as the cell suspensions used in this study.     

13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation

The influence of microbial degradation on the ¹³C/¹²C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors αC for toluene degradation by Pseudomonas putida (1.0026 ± 0.00017), Thauera aromatica (1.0017 ± 0.00015), Geobacter metallireducens (1.0018 ± 0.00029) and the sulphate-reducing strain TRM1 (1.0017 ± 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar ¹³C/¹²C isotope fractionation factor (αC = 1.0015 ± 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil–hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a ¹³C/¹²C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions.     

Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools

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. Λ 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 Bss-containing 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 Λ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller Λ 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 α subunit of Bss, correlated with the observed differences in Λ 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.     

Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product

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.     

Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase

The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, which is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radical-containing family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on Vmax and Vmax/Km when deuterated toluene is the substrate. At 30 degrees C the isotope effects are 1.7 +/- 0.2 and 2.9 +/- 0.1 on Vmax and Vmax/Km, respectively; at 4 degrees C they increase slightly to 2.2 +/- 0.2 and 3.1 +/- 0.1, respectively. We compare these results with the theoretical isotope effects on Vmax and Vmax/Km that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory [Himo, F. (2002) J. Phys. Chem. B 106, 7688-7692]. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.     

Co-metabolic conversion of toluene in anaerobic n-alkane-degrading bacteria

Diverse microorganisms have been described to degrade petroleum hydrocarbons anaerobically. Strains able to utilize n-alkanes do not grow with aromatic hydrocarbons, whereas strains able to utilize aromatic hydrocarbons do not grow with n-alkanes. To investigate this specificity in more detail, three anaerobic n-alkane degraders (two denitrifying, one sulfate-reducing) and eight anaerobic alkylbenzene degraders (five denitrifying, three sulfate-reducing) were incubated with mixtures of n-alkanes and toluene. Whereas the toluene degradationers formed only the characteristic toluene-derived benzylsuccinate and benzoate, but no n-alkane-derived metabolites, the n-alkane degraders formed toluene-derived benzylsuccinate, 4-phenylbutanoate, phenylacetate and benzoate besides the regular n-alkane-derived (1-methylalkyl)succinates and methyl-branched alkanoates. The co-metabolic conversion of toluene by anaerobic n-alkane degraders to the level of benzoate obviously follows the anaerobic n-alkane degradation pathway with C-skeleton rearrangement and decarboxylation rather than the β-oxidation pathway of anaerobic toluene metabolism. Hence, petroleum-derived aromatic metabolites detectable in anoxic environments may not be exclusively formed by genuine alkylbenzene degraders. In addition, the hitherto largely unexplored fate of fumarate hydrogen during the activation reactions was examined with (2,3-(2) H(2) )fumarate as co-substrate. Deuterium was completely exchanged with hydrogen at the substituted carbon atom (C-2) of the succinate adducts of n-alkanes, whereas it is retained in toluene-derived benzylsuccinate, regardless of the type of enzyme catalysing the fumarate addition reaction.     

Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors () of −1.5 and −3.9‰, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic (_intrinsic) were calculated. A comparison of _intrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average if no fractionation factor is available for single compounds.     

Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T.

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.     

H/D isotope effects on protein hydration and interaction in solution

An approach has been suggested to study the H/D isotope effect on protein-water and protein-protein intermolecular interactions by determining the content of non-freezing water using low-temperature (1)H NMR in mixed (H2O/D2O) water solutions. Direct data are obtained on the amount of H2O adsorbed (absolute hydration) in presence of the heavy isotope (deuterium D), and isothermals of H2O/D2O fractionation at protein surface groups are presented for temperatures between -10 degrees C and -35 degrees C and solutions of varying composition. The fractionation factor, phi = [x/(1 - x)]/[x(0)/(1 - x(0))], where x and x(0) are the fractions of deuterons in hydration and bulk water, respectively, appeared to be extremely high: phi > 1 at 0.03 < x(0) < 0.10. The high values of phi indicate a decrease in apparent hydration of protein molecules. A probable reason of the effect can be an inter-protein molecular solvent-mediated interaction induced by D2O. The excess of phi over 1 appears to provide a quantitative estimate of the fraction of hydration water affected by such interaction.     

Deuterium isotope effects on toluene metabolism. Product release as a rate-limiting step in cytochrome P-450 catalysis

Liver microsomes from phenobarbital-induced rats oxidize toluene to a mixture of benzyl alcohol plus o-, m- and p-cresol (ca. 69:31). Stepwise deuteration of the methyl group causes stepwise decreases in the yield of benzyl alcohol relative to cresols (ca. 24:76 for toluene-d3). For benzyl alcohol formation from toluene-d3 DV = 1.92 and D(V/K) = 3.53. Surprisingly, however, stepwise deuteration induces stepwise increases in total oxidation, giving rise to an inverse isotope effect overall (DV = 0.67 for toluene-d3). Throughout the series (i.e. d0, d1, d2, d3) the ratios of cresol isomers remain constant. These results are interpreted in terms of product release for benzyl alcohol being slower than release of cresols (or their epoxide precursors), and slow enough to be partially rate-limiting in turnover. Thus metabolic switching to cresol formation causes a net acceleration of turnover.     

Operon structure and expression of the genes for benzylsuccinate synthase in<Emphasis Type="Italic"> Thauera aromatica</Emphasis> strain K172

The first step in anaerobic toluene degradation is the addition of a fumarate cosubstrate to the methyl group of toluene, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme implicated in activating the enzyme by introducing the glycyl radical (BssD). Quantitation of the amounts of benzylsuccinate synthase and activating enzyme showed that both proteins are only synthesized in toluene-grown cells, and that the activating enzyme is present in about 14-fold lower amounts. Two mRNA species of the bss gene cluster were identified, one beginning in front of bssD, and a second in front of bssC. Only the first mRNA 5'-end correlates with a toluene-induced promoter, which is similar to that preceding the bbs operon coding for the further enzymes of toluene catabolism of the same strain. The second mapped 5'-end appears to be generated by endonucleolytic processing. The mRNA segment containing the bssD gene is very short-lived, while that containing the bssCAB genes is more stable. The RNA stability data are consistent with the observed amounts of encoded gene products. Furthermore, the previously known bssDCAB genes are apparently cotranscribed with a fifth gene ( bssE) whose product may function as a putative ATP-dependent chaperone for assembly and/or activation of benzylsuccinate synthase.     

Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.     

Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism

The C alpha primary hydrogen kinetic isotope effects (C alpha-KIEs) for the reaction of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with [alpha-2H]-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 +/- 0.03 and DV/KAsp = 1.36 +/- 0.04 in H2O; DV = 1.44 +/- 0.01 and DV/KAsp = 1.61 +/- 0.06 in D2O). The D2O solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C alpha (D2OV = 2.21 +/- 0.07 and D2OV/KAsp = 1.70 +/- 0.03 with [alpha-1H]-L-aspartate; D2OV = 2.34 +/- 0.12 and D2OV/KAsp = 1.82 +/- 0.06 with [alpha-2H]-L- aspartate). The C alpha-KIEs on V and on V/KAsp are independent of pH from pH 5.0 to pH 10.0. These results support a rate-determining concerted 1,3 prototropic shift mechanism by the multiple KIE criteria [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. The large C alpha-KIEs for the reaction of mitochondrial AATase (mAATase) with L-glutamate (DV = 1.88 +/- 0.13 and DV/KGlu = 3.80 +/- 0.43 in H2O; DV = 1.57 +/- 0.05 and DV/KGlu = 4.21 +/- 0.19 in D2O) coupled with the relatively small SKIEs (D2OV = 1.58 +/- 0.04 and D2OV/KGlu = 1.25 +/- 0.05 with [alpha-1H]-L-glutamate; D2OV = 1.46 +/- 0.06 and D2OV/KGlu = 1.16 +/- 0.05 with [alpha-2H]-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Benzylfumaric, benzylmaleic, and Z- and E-phenylitaconic acids: synthesis, characterization, and correlation with a metabolite generated by Azoarcus tolulyticus Tol-4 during anaerobic toluene degradation.

E-Phenylitaconic acid has been isolated as a metabolite generated by Azoarcus tolulyticus Tol-4 along with benzylsuccinic acid during anaerobic degradation of toluene. Strain Tol-4 converted 1 to 2% of toluene carbon to E-phenylitaconate and benzylsuccinate (10:1). The identification of E-phenylitaconic acid was based on 1H nuclear magnetic resonance (NMR) characterization of degradation products derived from 13C-labeled toluene followed by comparison of spectroscopic and chromatographic data for the isolated, unlabeled metabolite with those for chemically synthesized benzylfumaric acid, benzylmaleic acid, E-phenylitaconic acid, and Z-phenylitaconic acid. Spectroscopic comparisons included 1H NMR, 13C NMR, and nuclear overhauser effect correlations. High-pressure liquid chromatography (HPLC) retention times and HPLC coinjections with synthetic dioic acids provided another reliable line of evidence for structure assignment. The formation of E-phenylitaconic acid differs from previous reports of benzylfumaric acid generation along with benzylsuccinic acid during anaerobic microbial degradation of toluene. This has important implications relevant to elaboration of the metabolic route for anaerobic toluene degradation by strain Tol-4 and related organisms. Similar amounts of E-phenylitaconic acid were also produced by seven other strains of A. tolulyticus.     

Unusual four-bond secondary H/D isotope effect supports a short-strong hydrogen bond between phospholipase A2 and a transition state analogue inhibitor

A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA(2) in the presence of a phosphonate transition state analogue. The cross-modulation of H(epsilon2)/H48 and H(delta1)/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H(2)O/D(2)O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA(2), the observation that H(delta1)/H48 induces a 3-fold larger H/D secondary isotope effect ( approximately 0.15 ppm) on H(epsilon2)/H48 than vice versa ( approximately 0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short-strong hydrogen bond formed between the donor N(delta1)/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA(2) and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.     

Linking low-level stable isotope fractionation to expression of the cytochrome P450 monooxygenase-encoding ethB gene for elucidation of methyl tert-butyl ether biodegradation in aerated treatment pond systems

Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ε(C)] of -0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ε(H)]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (ε(C) of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ε(H) of -5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem.