160 results on '"Hans-Peter E. Kohler"'
Search Results
2. Biodegradation of poly(butylene succinate) in soil laboratory incubations assessed by stable carbon isotope labelling
- Author
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Taylor F. Nelson, Rebekka Baumgartner, Madalina Jaggi, Stefano M. Bernasconi, Glauco Battagliarin, Carsten Sinkel, Andreas Künkel, Hans-Peter E. Kohler, Kristopher McNeill, and Michael Sander
- Subjects
Science - Abstract
This study applies stable carbon isotope labelling to study polymer biodegradation in soils. This labelling enables accurate and precise tracking of polymer carbon during biodegradation and, thereby, provides a holistic picture of this process.
- Published
- 2022
- Full Text
- View/download PDF
3. Enzyme Kinetics of Organic Contaminant Oxygenations
- Author
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Charlotte E. Bopp, Hans-Peter E. Kohler, and Thomas B. Hofstetter
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biodegradation ,catalytic cycles ,non-heme fe ii oxygenases ,Chemistry ,QD1-999 - Abstract
Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O2, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O2 uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O2 activation to substrate hydroxylation, depending on the extent of O–O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O2 uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively.
- Published
- 2020
- Full Text
- View/download PDF
4. ipso-Substitution – A Novel Pathway for Microbial Metabolism of Endocrine-Disrupting 4-Nonylphenols, 4-Alkoxyphenols, and Bisphenol A
- Author
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Hans-Peter E. Kohler, Frédéric L. P. Gabriel, and Walter Giger
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Alkoxyphenols ,Bisphenol a ,Endocrine disruptors ,Nonylphenol ,Sphingobium xenophagum bayram ,Ipso-substitution ,Chemistry ,QD1-999 - Abstract
Our studies with Sphingobium xenophagum Bayram show that this bacterial strain degrades ?-quaternary 4-nonylphenols by an ipso-substitution mechanism, whereby the nonylphenol substrates are initially hydroxylated at the ipso position to form 4-hydroxy-4-nonylcyclohexa-2,5-dienones (quinols). Subsequently, the ?-quaternary side chains are able to detach as short-living cations from these intermediates. Alkyl branches attached to the carbocation help to delocalize and thereby stabilize the positive charge through inductive and hyperconjugative effects, which explains why only alkyl moieties of ?-quaternary nonylphenols are released. This view is corroborated by experiments with S. xenophagum Bayram, in which the alkyl chains of the non-?-quaternary 4-(1-methyloctyl) phenol (4-NP2) and 4-n-nonylphenol (4-NP1) were not released, so that the bacterium was unable to utilize these isomers as growth substrates. Analysis of dead end metabolites and experiments with 18O labeled H2O and O2 clearly show that in the main degradation pathway the nonyl cation derived from ?-quaternary quinols preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the incorporation of significant amounts of O2-derived oxygen into the nonanol metabolites derived from degradation of certain ?,?-dimethyl substituted nonylphenols by strain Bayram strongly indicates the existence of a minor pathway in which the cation undergoes an alternative reaction and attacks the ipso-hydroxy group, yielding a 4-alkoxyphenol as an intermediate. Additional growth experiments with strain Bayram revealed that also the two alkoxyphenols 4-tert-butoxyphenol and 4-n-octyloxyphenol promote growth. Furthermore, strain Bayram's ipso-hydroxlating activity is able to transform also bisphenol A.
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- 2008
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- View/download PDF
5. Occurrence and Fate of Antibiotics as Trace Contaminants in Wastewaters, Sewage Sludges, and Surface Waters
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Walter Giger, Alfredo C. Alder, Eva M. Golet, Hans-Peter E. Kohler, Christa S. McArdell, Eva Molnar, Hansrudolf Siegrist, and Marc J.-F. Suter
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Ciprofloxacin ,Clarithromycin ,Fluoroquinolones ,Macrolides ,Norfloxacin ,Chemistry ,QD1-999 - Abstract
Environmental analytical studies show that trace concentrations of antibacterial agents (antibiotics) occur in hospital and municipal wastewaters and in the aquatic environment. Fluoroquinolones and macrolides, two important human-use antibiotic classes, were studied in detail. The results are discussed regarding input sources and behavior in wastewater treatment and rivers. The fluoroquinolones ciprofloxacin and norfloxacin are substantially eliminated in wastewater treatment (80–90%) by sorption transfer to sewage sludge. In digested sludges the fluoroquinolones occur at mg/kg levels. Ciprofloxacin and norfloxacin are further removed in the Glatt river by 66 and 48%, respectively. The most abundant macrolide clarithromycin was detected at 57 to 330 ng/l concentrations in treated wastewater effluents. Different compositions of the macrolides (clarithromycin and erythromycin-H2O) determined in treated effluents of three wastewater treatment plants can be explained by distinct consumption patterns, in one case due to an international airport located in the catchment area. Residual levels of clarithromycin in the Glatt river were up to 75 ng/l with no apparent removal in the river. These results provide important information on environmental exposures, which can be incorporated into environmental risk assessments of the particular chemicals.
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- 2003
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6. Environmental Fate of Chiral Pollutants &ndash the Necessity of Considering Stereochemistry
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Hans-Peter E. Kohler, Werner Angst, Walter Giger, Carlo Kanz, Stephan Müller, and Marc J.-F. Suter
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Chemistry ,QD1-999 - Abstract
Many organic compounds regulated by environmental laws are chiral and are released into the environment as racemates. 3-Phenylbutanoic acid and mecoprop, two chiral pollutants, were enantioselectively degraded by pure cultures of microorganisms. This indicates the importance of assessing the environmental impact of stereoisomers separately, because selective enrichment of one of the enantiomers may occur in the environment. Field studies on the fate of highly polar, chiral compounds, like sulfophenylcarboxylates, are hampered by the lack of appropriate analytical methods for the separation of the enantiomers. Therefore, a method based on capillary electrophoresis with ?-cyclodextrin as chiral selector was developed to separate the enantiomers of such compounds. In a field study at a Swiss waste disposal site, the fate of the chiral herbicide mecoprop was investigated. The enantiomeric ratio of (R)-mecoprop to (S)-mecoprop altered during groundwater passage of landfill leachate. This is a strong indication for in situ biodegradation. Our data imply that not only the enantiomers of a chiral drug or pesticide may exert different effects on the biological targets, but also their biodegradation and environmental fate may differ.
- Published
- 1997
7. Substrate-Specific Coupling of O2 Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases
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Sarah G. Pati, Charlotte E. Bopp, Hans-Peter E. Kohler, and Thomas B. Hofstetter
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General Chemistry ,Catalysis - Published
- 2022
8. Elucidating the Role of O
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Charlotte E, Bopp, Nora M, Bernet, Hans-Peter E, Kohler, and Thomas B, Hofstetter
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Oxygenations of aromatic soil and water contaminants with molecular O
- Published
- 2022
9. Quantification of Synthetic Polyesters from Biodegradable Mulch Films in Soils
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Kristopher McNeill, Stephanie Christa Remke, Hans-Peter E. Kohler, Taylor F. Nelson, and Michael Sander
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Chloroform ,Polyesters ,Extraction (chemistry) ,Temperature ,Agriculture ,Deuterated chloroform ,General Chemistry ,010501 environmental sciences ,Biodegradation ,01 natural sciences ,Polyester ,Soil ,chemistry.chemical_compound ,Biodegradation, Environmental ,chemistry ,Polylactic acid ,Soil water ,Environmental Chemistry ,Methanol ,0105 earth and related environmental sciences ,Nuclear chemistry - Abstract
Soil biodegradable mulch films composed of the polyester polybutylene adipate-co-terephthalate (PBAT) are being increasingly used in agriculture. Analytical methods to quantify PBAT in field soils are needed to assess its soil occurrence and fate. Here, we report an analytical method for PBAT in soils that couples Soxhlet extraction or accelerated solvent extraction (ASE) with quantitative protonnuclear magnetic resonance (q-1H NMR) spectroscopy detection. The 1H NMR peak areas of aromatic PBAT protons increased linearly with PBAT concentrations dissolved in deuterated chloroform (CDCl3), demonstrating accurate quantitation of PBAT by q-1H NMR. Spike-recovery experiments involving PBAT addition to model sorbents and soils showed increased PBAT extraction efficiencies into chloroform (CHCl3) with methanol (MeOH) as cosolvent, consistent with MeOH competitively displacing PBAT from H-bond donating sites on mineral surfaces. Systematic variations in solvent composition and temperatures in ASE revealed quantitative PBAT extraction from soil with 90/10 volume % CHCl3/MeOH at 110-120 °C. Both Soxhlet extraction and ASE resulted in the complete recovery of PBAT added to a total of seven agricultural soils covering a range of physicochemical properties, independent of whether PBAT was added to soils dissolved in CHCl3, as film, or as particles. Recovery was also complete for PBAT added to soil in the form of a commercial soil biodegradable mulch film with coextractable polylactic acid (PLA). The presented analytical method enables accurate quantification and biodegradation monitoring of PBAT in agricultural field soils.
- Published
- 2019
10. Enzymatic synthesis and formation kinetics of mono- and di-hydroxylated chlorinated paraffins with the bacterial dehalogenase LinB from Sphingobium indicum
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Norbert V. Heeb, Davide Bleiner, Hans-Peter E. Kohler, Susanne Kern, Flurin Mathis, Marco C. Knobloch, and Thomas Fleischmann
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Environmental Engineering ,Halogenation ,Dihydroxylated chloroparaffins (CP-diols) ,Health, Toxicology and Mutagenesis ,Kinetics ,In-vitro CP transformation ,Hydroxylated chloroparaffins (CP-ols) ,Hydroxylation ,chemistry.chemical_compound ,Reaction rate constant ,Chlorinated paraffins ,Hydrocarbons, Chlorinated ,polycyclic compounds ,Humans ,Environmental Chemistry ,Organic chemistry ,Persistent organic pollutants (POPs) ,Dehalogenase ,chemistry.chemical_classification ,Public Health, Environmental and Occupational Health ,General Medicine ,General Chemistry ,Pollution ,540: Chemie ,Sphingomonadaceae ,Transformation (genetics) ,Enzyme ,chemistry ,Paraffin ,Enzymatic dehalohydroxylation ,Environmental Monitoring ,Sphingobium indicum - Abstract
Transformation studies of chlorinated paraffins (CPs) and the effects of CP transformation products on humans, biota and environment are rare. The focus here is on hydroxylation reactions. As for polyhalogenated persistent organic pollutants (POPs) in general, hydroxylation reactions convert lipophilic material to more polar compounds with increased mobility. We investigated the in-vitro transformation of single-chain CP-mixtures to hydroxylated products with the dehalogenase LinB from Sphingobium indicum. C11-, C12- and C13-single-chain CP-homologues were exposed to LinB and mono-hydroxylated (CP-ols) and di-hydroxylated (CP-diols) transformation products were formed. Liquid-chromatography coupled to mass-spectrometry (LC-MS) was used to detect hydroxylated products and separate them from the starting material. The presented data can be used to identify these CP-ol and CP-diol homologues in other samples. Hydroxylated products had lower chlorination degrees (nCl) than respective CP-starting-materials. Reactive and persistent CP-material was found in each homologue group. Reactive material is converted within hours by LinB, while more persistent CPs are transformed within days. Homologue-specific kinetic models were established to simulate the stepwise hydroxylation of persistent CPs to mono- and di-hydroxylated products. First-order rate constants for the formation of CP-ols (k1) and CP-diols (k2) were deduced for different homologues. Lower-chlorinated CP-ols did not accumulate to large extent and were transformed quickly to CP-diols, while higher-chlorinated CP-ols and -diols both accumulated. By enzymatic transformation of single-chain CPs with LinB, we synthesized unique sets of mono- and di-hydroxylated materials, which can be used as analytical standards and as starting materials for metabolic, toxicity and environmental fate studies.
- Published
- 2021
11. Biodegradation of poly(butylene succinate) in soil laboratory incubations assessed by stable carbon isotope labelling
- Author
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Taylor F, Nelson, Rebekka, Baumgartner, Madalina, Jaggi, Stefano M, Bernasconi, Glauco, Battagliarin, Carsten, Sinkel, Andreas, Künkel, Hans-Peter E, Kohler, Kristopher, McNeill, and Michael, Sander
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Carbon Isotopes ,Soil ,Biodegradation, Environmental ,Polymers ,Isotope Labeling ,Polyesters ,Succinates ,Carbon Dioxide ,Butylene Glycols ,Plastics ,Carbon - Abstract
Using biodegradable instead of conventional plastics in agricultural applications promises to help overcome plastic pollution of agricultural soils. However, analytical limitations impede our understanding of plastic biodegradation in soils. Utilizing stable carbon isotope (
- Published
- 2021
12. Assessing Aerobic Biotransformation of Hexachlorocyclohexane Isomers by Compound-Specific Isotope Analysis
- Author
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Iris E. Schilling, Charlotte E. Bopp, Thomas B. Hofstetter, Rup Lal, and Hans-Peter E. Kohler
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Halogenation ,Isotope ,Hexachlorocyclohexane ,Substrate (chemistry) ,General Chemistry ,010501 environmental sciences ,Biodegradation ,01 natural sciences ,chemistry.chemical_compound ,Biodegradation, Environmental ,Isotope fractionation ,Isomerism ,chemistry ,Biotransformation ,Environmental chemistry ,Kinetic isotope effect ,Environmental Chemistry ,0105 earth and related environmental sciences ,Isotope analysis - Abstract
Contamination of soils and sediments with the highly persistent hexachlorocyclohexanes (HCHs) continues to be a threat for humans and the environment. Despite the existence of bacteria capable of biodegradation and cometabolic transformation of HCH isomers, such processes occur over time scales of decades and are thus challenging to assess. Here, we explored the use of compound-specific isotope analysis (CSIA) to track the aerobic biodegradation and biotransformation pathways of the most prominent isomers, namely, (-)-α-, (+)-α-, β-, γ-, and δ-HCH, through changes of their C and H isotope composition in assays of LinA2 and LinB enzymes. Dehydrochlorination of (+)-α-, γ-, and δ-HCH catalyzed by LinA2 was subject to substantial C and H isotope fraction with apparent 13C- and 2H-kinetic isotope effects (AKIEs) of up to 1.029 ± 0.001 and 6.7 ± 2.9, respectively, which are indicative of bimolecular eliminations. Hydrolytic dechlorination of δ-HCH by LinB exhibited even larger C but substantially smaller H isotope fractionation with 13C- and 2H-AKIEs of 1.073 ± 0.006 and 1.41 ± 0.04, respectively, which are typical for nucleophilic substitutions. The systematic evaluation of isomer-specific phenomena showed that, in addition to contaminant uptake limitations, diffusion-limited turnover ((-)-α-HCH), substrate dissolution (β-HCH), and potentially competing reactions catalyzed by constitutively expressed enzymes might bias the assessment of HCH biodegradation by CSIA at contaminated sites.
- Published
- 2019
13. Kinetic Isotope Effects of the Enzymatic Transformation of γ-Hexachlorocyclohexane by the Lindane Dehydrochlorinase Variants LinA1 and LinA2
- Author
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Iris E. Schilling, Hans-Peter E. Kohler, Ramon Hess, Jakov Bolotin, Thomas B. Hofstetter, and Rup Lal
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chemistry.chemical_classification ,Chemistry ,Stereochemistry ,Stable isotope ratio ,Lyases ,General Chemistry ,Fractionation ,010501 environmental sciences ,01 natural sciences ,Kinetics ,chemistry.chemical_compound ,Isotope fractionation ,Enzyme ,Isotopes ,Kinetic isotope effect ,Environmental Chemistry ,Lindane ,Conformational isomerism ,Hexachlorocyclohexane ,0105 earth and related environmental sciences ,Isotope analysis - Abstract
Compound-specific isotope analysis (CSIA) can provide insights into the natural attenuation processes of hexachlorocyclohexanes (HCHs), an important class of persistent organic pollutants. However, the interpretation of HCH stable isotope fractionation is conceptually challenging. HCHs exist as different conformers that can be converted into each other, and the enzymes responsible for their transformation discriminate among those HCH conformers. Here, we investigated the enzyme specificity of apparent 13C- and 2H-kinetic isotope effects (AKIEs) associated with the dehydrochlorination of γ-HCH (lindane) by two variants of the lindane dehydrochlorinases LinA1 and LinA2. While LinA1 and LinA2 attack γ-HCH at different trans-1,2-diaxial H–C–C–Cl moieties, the observed C and H isotope fractionation was large, typical for bimolecular eliminations, and was not affected by conformational mobility. 13C-AKIEs for transformation by LinA1 and LinA2 were the same (1.024 ± 0.001 and 1.025 ± 0.001, respectively), wherea...
- Published
- 2019
14. Transformation of short-chain chlorinated paraffins and olefins with the bacterial dehalogenase LinB from Sphingobium Indicum - Kinetic models for the homologue-specific conversion of reactive and persistent material
- Author
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Susanne Kern, Flurin Mathis, Lena Schinkel, Marco C. Knobloch, Davide Bleiner, Norbert V. Heeb, and Hans-Peter E. Kohler
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Environmental Engineering ,Health, Toxicology and Mutagenesis ,0208 environmental biotechnology ,02 engineering and technology ,010501 environmental sciences ,Alkenes ,01 natural sciences ,Catalysis ,Chlorinated paraffins ,polycyclic compounds ,Hydrocarbons, Chlorinated ,Environmental Chemistry ,Organic chemistry ,Reactivity (chemistry) ,Persistent organic pollutants (POPs) ,Chlorinated olefins (COs) ,0105 earth and related environmental sciences ,Dehalogenase ,Chemistry ,Public Health, Environmental and Occupational Health ,Halogenation ,General Medicine ,General Chemistry ,Pollution ,First-order kinetic model ,660: Technische Chemie ,020801 environmental engineering ,Sphingomonadaceae ,Kinetics ,Paraffin ,Chlorinated paraffins (CPs) ,Enzymatic dechlorination ,Reactive material ,Haloalkane dehalogenase ,Sphingobium indicum ,Environmental Monitoring - Abstract
Structure, reactivity and physico-chemical properties of polyhalogenated compounds determine their up-take, transport, bio-accumulation, transformation and toxicity and their environmental fate. In technical mixtures of chlorinated paraffins (CPs), these properties are distributed due to the presence of thousands of homologues. We hypothesized that roles of CP dehalogenation reactions, catalyzed by the haloalkane dehalogenase LinB, depend on structural properties of the substrates, e.g. chlorination degree and carbon-chain length. We exposed mixtures of chlorinated undecanes, dodecanes and tridecanes in-vitro to LinB from Sphingobium Indicum bacteria. These single-chain CP-materials also contain small amounts of chlorinated olefins (COs), which can be distinct by mathematical deconvolution of respective mass-spectra. With this procedure, we obtained homologue-specific transformation kinetics of substrates differing in saturation degree, chlorination degree and carbon chain-length. For all homologues, two-stage first-order kinetic models were established, which described the faster conversion of reactive material and the slower transformation of more persistent material. Half-lifes of 0.5–3.2 h and 56–162 h were determined for more reactive and more persistent CP-material. Proportions of persistent material increased steadily from 18 to 67% for lower (Cl6) to higher (Cl11) chlorinated paraffins and olefins. Conversion efficiencies decreased with increasing chlorination degree from 97 to 70%. Carbon-chain length had only minor effects on transformation rates. Hence, the conversion was faster and more efficient for lower-chlorinated material, and slower for higher-chlorinated and longer-chained CPs and COs. Current legislation has banned short-chain chlorinated paraffins (SCCPs) and forced a transition to longer-chain CPs. This may be counterproductive with regard to enzymatic transformation with LinB.
- Published
- 2021
15. Transformation of short-chain chlorinated paraffins by the bacterial haloalkane dehalogenase LinB : Formation of mono- and di-hydroxylated metabolites
- Author
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Marco C. Knobloch, Peter Lienemann, Norbert V. Heeb, Iris Schilling, Hans-Peter E. Kohler, Davide Bleiner, and Lena Schinkel
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Environmental Engineering ,Hydroxylated chlorinated paraffins ,Halogenation ,Hydrolases ,Health, Toxicology and Mutagenesis ,0208 environmental biotechnology ,02 engineering and technology ,010501 environmental sciences ,medicine.disease_cause ,Hydroxylation ,01 natural sciences ,Chloride ,Adduct ,Chlorinated paraffins ,Liquid chromatography–mass spectrometry ,medicine ,Escherichia coli ,Hydrocarbons, Chlorinated ,Environmental Chemistry ,Organic chemistry ,Persistent organic pollutants (POPs) ,Haloalkane dehalogenase LinB ,Chlorinated olefins (COs) ,0105 earth and related environmental sciences ,Chemistry ,Public Health, Environmental and Occupational Health ,General Medicine ,General Chemistry ,Pollution ,660: Technische Chemie ,020801 environmental engineering ,Hydrocarbons, Brominated ,Sphingomonadaceae ,Paraffin ,Chlorinated paraffins (CPs) ,Mass spectrum ,Biocatalysis ,Environmental Pollutants ,Heterologous expression ,Hexachlorocyclohexane ,Haloalkane dehalogenase ,medicine.drug ,Environmental Monitoring - Abstract
Short-chain chlorinated paraffins (SCCPs) are listed as persistent organic pollutants (POPs) under the Stockholm Convention. Such substances are toxic, bioaccumulating, transported over long distances and degrade slowly in the environment. Certain bacterial strains of the Sphingomonadacea family are able to degrade POPs, such as hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). The haloalkane dehalogenase LinB, expressed in certain Sphingomonadacea, is able to catalyze the transformation of haloalkanes to hydroxylated compounds. Therefore, LinB is a promising candidate for conversion of SCCPs. Hence, a mixture of chlorinated tridecanes was exposed in vitro to LinB, which was obtained through heterologous expression in Escherichia coli. Liquid chromatography mass spectrometry (LC-MS) was used to analyze chlorinated tridecanes and their transformation products. A chloride-enhanced soft ionization method, which favors the formation of chloride adducts [M+Cl]- without fragmentation, was applied. Mathematical deconvolution was used to distinguish interfering mass spectra of paraffinic, mono-olefinic and di-olefinic compounds. Several mono- and di-hydroxylated products including paraffinic, mono-olefinic and di-olefinic compounds were found after LinB exposure. Mono- (rt = 5.9-6.9 min) and di-hydroxylated (rt = 3.2-4.5 min) compounds were separated from starting material (rt = 7.7-8.5 min) by reversed phase LC. Chlorination degrees of chlorinated tridecanes increased during LinB-exposure from nCl = 8.80 to 9.07, indicating a preferential transformation of lower chlorinated (Cl
- Published
- 2021
16. Kinetics and stereochemistry of LinB-catalyzed δ-HBCD transformation: Comparison of in vitro and in silico results
- Author
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Hans-Peter E. Kohler, Norbert V. Heeb, Manuel Mazenauer, Simon Wyss, Birgit Geueke, and Peter Lienemann
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0301 basic medicine ,Environmental Engineering ,Stereochemistry ,Health, Toxicology and Mutagenesis ,Kinetics ,010501 environmental sciences ,01 natural sciences ,Michaelis–Menten kinetics ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Bromide ,Environmental Chemistry ,Computer Simulation ,Enzyme kinetics ,0105 earth and related environmental sciences ,Chemistry ,Public Health, Environmental and Occupational Health ,Stereoisomerism ,General Medicine ,General Chemistry ,Pollution ,Hydrocarbons, Brominated ,Enzyme binding ,030104 developmental biology ,Docking (molecular) ,Enantiomer ,Haloalkane dehalogenase - Abstract
LinB is a haloalkane dehalogenase found in Sphingobium indicum B90A, an aerobic bacterium isolated from contaminated soils of hexachlorocyclohexane (HCH) dumpsites. We showed that this enzyme also converts hexabromocyclododecanes (HBCDs). Here we give new insights in the kinetics and stereochemistry of the enzymatic transformation of δ-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P1δ, P2δ) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T1δ, T2δ). Enzymatic transformations of δ-HBCD, α1-PBCDol, one of the transformation products, and α2-PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters KM, vmax, kcat/KM indicated that δ-HBCD is the best LinB substrate followed by α2- and α1-PBCDol. The stereochemistry of these transformations was modeled in silico, investigating respective enzyme-substrate (ES) and enzyme-product (EP) complexes. One of the four predicted ES-complexes led to the PBCDol product P1δ, identical to α2-PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An SN2-like substitution of bromine at C6 of δ-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to α2-PBCDol. Modeling results further indicate that backside attacks at C1, C9 and C10 are reasonable too, selectively binding leaving bromide ions in a halide pocket found in LinB. Docking with α2-PBCDol, also allowed productive enzyme binding. A TBCD-1,5-diol with the 1S,2S,5R,6R,9S,10R-configuration is the predicted second-generation product T1δ. In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of δ-HBCD to specific PBCDols and TBCDdiols at A-resolution and predictions of their stereochemistry.
- Published
- 2018
17. Effect of Metabolic Constraints on the Observable Isotope Fractionation Associated with Aerobic Biodegradation of Hexachlorocyclohexanes
- Author
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Hans-Peter E. Kohler, Iris E. Schilling, and Thomas B. Hofstetter
- Subjects
chemistry.chemical_classification ,Isotope fractionation ,Enzyme ,Haloalkane ,chemistry ,Isotope ,Environmental chemistry ,Kinetic isotope effect ,Biodegradation ,Contamination ,Isotope analysis - Abstract
Biodegradation of the highly persistent hexachlorocyclohexanes is difficult to assess in contaminated soils and sediments because this process occurs only slowly over timescales of years to decades. Recent instrumental advances for compound-specific isotope analysis (CSIA) now make it possible to monitor such processes based on the isotope fractionation of multiple elements as changes of 13C/12C, 37Cl/35Cl, and 2H/1H ratios in the residual HCH contamination. However, metabolic constraints from the expression of enzymes that can compete with the primary metabolic reactions leading to HCH biodegradation and thus alter the observable contaminant isotope fractionation through co metabolic side reactions have largely been overlooked. Here, we developed activity-based assays to assess the competitive behaviour of mixtures of lindane dehydrochlorinase LinA and haloalkane dehydrochlorinase LinB which catalyze the dehydrochlorination and hydrolytic dechlorination of several HCH isomers. Using X-HCH as model contaminant that can be transformed by both enzymes in mixtures of different LinA2/LinB activity, we observed preferential formation of products from hydrolytic dechlorination. This observation suggests that LinB was more reactive than predicted from the nominal enzyme activities. The C and H isotope fractionation of X-HCH in LinA2/LinB mixtures can be rationalized by a combination of isotope enrichment factors from independent dehydrochlorination and hydrolytic dechlorination reactions where LinA2 contributed more to H isotope fractionation than LinB, thus contrasting the assessment of competitive enzyme activity. Our study shows that metabolic constraints associated with the expression of multiple enzymes can potentially compromise inferences of extent and pathways of contaminant biodegradation from CSIA.
- Published
- 2019
18. Assessing the environmental transformation of nanoplastic through 13C-labelled polymers
- Author
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Michael Sander, Kristopher McNeill, and Hans-Peter E. Kohler
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chemistry.chemical_classification ,Materials science ,Biomedical Engineering ,Bioengineering ,Nanotechnology ,02 engineering and technology ,Polymer ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,01 natural sciences ,Atomic and Molecular Physics, and Optics ,Transformation (music) ,0104 chemical sciences ,chemistry ,General Materials Science ,Electrical and Electronic Engineering ,0210 nano-technology - Abstract
To assess potential risks posed by plastic nanoparticles, we must study the way in which they transfer and transform in the environment. Using 13C-labelled nanoplastics could provide a safe and effective way to establish whether the plastic is mineralized or whether it persists in the environment.
- Published
- 2019
19. Dos and Do Nots When Assessing the Biodegradation of Plastics
- Author
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Michael Sander, Kristopher McNeill, Hans-Peter E. Kohler, Ramani Narayan, and Michael T. Zumstein
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Biodegradation, Environmental ,Waste management ,Chemistry ,MEDLINE ,Environmental Chemistry ,General Chemistry ,Biodegradation ,Plastics - Published
- 2019
20. Aminobacter sp. MSH1 mineralises the groundwater micropollutant 2,6-dichlorobenzamide through a unique chlorobenzoate catabolic pathway
- Author
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Benjamin Horemans, Hans-Peter E. Kohler, Ruddy Wattiez, René De Mot, Daniel Rentsch, Maarten G. K. Ghequire, Dirk Springael, Bart Raes, and Jeroen T’Syen
- Subjects
0303 health sciences ,Chlorobenzoates ,030306 microbiology ,Catabolism ,Chemistry ,Stereochemistry ,General Chemistry ,Monooxygenase ,6. Clean water ,Amidase ,Hydroxylation ,03 medical and health sciences ,chemistry.chemical_compound ,Bioremediation ,13. Climate action ,Hydrolase ,Environmental Chemistry ,030304 developmental biology ,Dehalogenase - Abstract
2,6-Dichlorobenzamide (BAM) is a major groundwater micropollutant posing problems for drinking water treatment plants (DWTPs) that depend on groundwater intake. Aminobacter sp. MSH1 uses BAM as the sole source of carbon, nitrogen, and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge of its BAM-catabolic pathway, which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the monooxygenase BbdD transforming 2,6-DCBA into 2,6-dichloro-3-hydroxybenzoic acid. Here, we show that the 2,6-DCBA catabolic pathway is unique and differs substantially from catabolism of other chlorobenzoates. BbdD catalyzes a second hydroxylation, forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyze the thiolytic removal of the first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the α/β hydrolase superfamily (BbdC). BbdC is the first enzyme in that superfamily associated with dehalogenation of chlorinated aromatics and appears to represent a new subtype within the α/β hydrolase dehalogenases. The activity of BbdC yields a unique trihydroxylated aromatic intermediate for ring cleavage that is performed by an extradiol dioxygenase (BbdF) producing 2,4,6-trioxoheptanedioic acid, which is likely converted to Krebs cycle intermediates by BbdG. ispartof: Environmental Science & Technology vol:53 issue:17 pages:10146-10156 ispartof: location:United States status: published
- Published
- 2019
21. Photochemical Transformation of Poly(butylene adipate- co-terephthalate) and Its Effects on Enzymatic Hydrolyzability
- Author
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Melissa A. Maurer-Jones, Marc A. Hillmyer, Isabelle Rüegsegger, Kristopher McNeill, Michael T. Zumstein, Michael Sander, Hans-Peter E. Kohler, Gordon J. Getzinger, and Guilhem X. De Hoe
- Subjects
chemistry.chemical_classification ,Materials science ,Adipates ,Polyesters ,Phthalic Acids ,General Chemistry ,Polymer ,010501 environmental sciences ,Biodegradation ,Alkenes ,Photochemistry ,01 natural sciences ,Polyester ,Phthalic acid ,chemistry.chemical_compound ,Hydrolysis ,chemistry ,Enzymatic hydrolysis ,Adipate ,Environmental Chemistry ,Thermal analysis ,0105 earth and related environmental sciences - Abstract
Biodegradable polyesters are being increasingly used to replace conventional, nondegradable polymers in agricultural applications such as plastic film for mulching. For many of these applications, poly(butylene adipate- co-terephthalate) (PBAT) is a promising biodegradable material. However, PBAT is also susceptible to photochemical transformations. To better understand how photochemistry affects the biodegradability of PBAT, we irradiated blown, nonstabilized, transparent PBAT films and studied their enzymatic hydrolysis, which is considered the rate-limiting step in polyester biodegradation. In parallel, we characterized the irradiated PBAT films by dynamic mechanical thermal analysis. The rate of enzymatic PBAT hydrolysis decreased when the density of light-induced cross-links within PBAT exceeded a certain threshold. Mass-spectrometric analysis of the enzymatic hydrolysis products of irradiated PBAT films provided evidence for radical-based cross-linking of two terephthalate units that resulted in the formation of benzophenone-like molecules. In a proof-of-principle experiment, we demonstrated that the addition of photostabilizers to PBAT films mitigated the negative effect of UV irradiation on the enzymatic hydrolyzability of PBAT. This work advances the understanding of light-induced changes on the enzyme-mediated hydrolysis of aliphatic-aromatic polyesters and will therefore have important implications for the development of biodegradable plastics.
- Published
- 2019
22. Modelling carbofuran biotransformation by: Novosphingobium sp. KN65.2 in the presence of coincidental carbon and indigenous microbes
- Author
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Damian E. Helbling, Hans-Peter E. Kohler, Li Liu, and Barth F. Smets
- Subjects
Bioconversion ,Environmental Engineering ,Aquatic environments ,Microorganism ,Microorganisms ,Microbial communities ,Biochemistry ,Carbon substrates ,chemistry.chemical_compound ,Biotransformation ,Batch experiments ,SDG 14 - Life Below Water ,Pesticides ,Biology ,Organic carbon ,Water Science and Technology ,Total organic carbon ,Assimilable organic carbon ,Biokinetic models ,Strain (chemistry) ,Organic Compounds ,Aquatic ecosystem ,Indigenous microbes ,Marine Science and Oceanography ,Additives ,Strain rate ,Natural waters ,Pesticide ,Chemical Agents and Basic Industrial Chemicals ,chemistry ,Aquatic organisms ,Environmental chemistry ,Carbofuran - Abstract
The influence that coincidental carbon substrates (i.e., assimilable organic carbon, AOC) and indigenous microbial communities has on pesticide biotransformation by degrader strains in aquatic environments is poorly understood. We conducted batch experiments to investigate carbofuran biotransformation by Novosphingobium sp. KN65.2 using four environmentally derived water samples with varying amounts and types of AOC and indigenous microbial communities. We designed experimental scenarios to explore the influence of AOC and indigenous microbial communities on the growth of strain KN65.2 and the biotransformation of carbofuran. Relevant kinetic parameters were estimated from simpler experiments, and used to predict the growth of strain KN65.2 and the biotransformation of carbofuran in more complex experiments with an additive biokinetic model. We found that an additive biokinetic model adequately predicts the growth of strain KN65.2 and the rate of carbofuran biotransformation in natural waters that support the growth of strain KN65.2. However, our model over-predicts the growth of strain KN65.2 and the rate of carbofuran biotransformation in low-AOC environments. Overall, our results define the scope within which additive biokinetic models can be used to predict pesticide biotransformation in the presence of coincidental carbon substrates and indigenous microbial communities.
- Published
- 2019
23. Transformation of ε-HBCD with the Sphingobium Indicum enzymes LinA1, LinA2 and LinATM, a triple mutant of LinA2
- Author
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Rup Lal, Norbert V. Heeb, Thomas Fleischmann, Jasmin Hubeli, Peter Lienemann, Hans-Peter E. Kohler, and Namita Nayyar
- Subjects
Environmental Engineering ,Stereochemistry ,Health, Toxicology and Mutagenesis ,0208 environmental biotechnology ,02 engineering and technology ,010501 environmental sciences ,01 natural sciences ,chemistry.chemical_compound ,Biotransformation ,Escherichia coli ,Environmental Chemistry ,Enzyme kinetics ,Flame Retardants ,0105 earth and related environmental sciences ,chemistry.chemical_classification ,Public Health, Environmental and Occupational Health ,Substrate (chemistry) ,Stereoisomerism ,General Medicine ,General Chemistry ,Pollution ,Hydrocarbons, Brominated ,020801 environmental engineering ,Amino acid ,Sphingomonadaceae ,Enzyme ,chemistry ,Enantiomer ,Lindane ,Hexachlorocyclohexane ,Sphingobium indicum - Abstract
Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, e-HBCD has the highest symmetry. This makes e-HBCD an interesting substrate to study the selectivity of biotransformations. We expressed three LinA dehydrohalogenase enzymes in E. coli bacteria, two wild-type, originating from Sphingobium indicum B90A bacteria and LinATM, a triple mutant of LinA2, with mutations of L96C, F113Y and T133 M. These enzymes are involved in the hexachlorocyclohexane (HCH) metabolism, specifically of the insecticide γ-HCH (Lindane). We studied the reactivity of those eight HBCD stereoisomers found in technical HBCD. Furthermore, we compared kinetics and selectivity of these LinA variants with respect to e-HBCD. LC-MS data indicate that all enzymes converted e-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants kcat and enzyme specificities kcat/KM indicate that e-HBCD conversion was fastest and most specific with LinA2. Only one PBCDen stereoisomer was formed by LinA2, while LinA1 and LinATM produced mixtures of two PBCDE enantiomers at three times lower rates than LinA2. In analogy to the biotransformation of (−)β-HBCD, with selective conversion of dibromides in R-S-configuration, we assume that 1E,5S,6R,9S,10R-PBCDen is the e-HBCD transformation product from LinA2. Implementing three amino acids of the LinA1 substrate-binding site into LinA2 resulted in a triple mutant with similar kinetics and product specificity like LinA1. Thus, point-directed mutagenesis is an interesting tool to modify the substrate- and product-specificity of LinA enzymes and enlarge their scope to metabolize other halogenated persistent organic pollutants regulated under the Stockholm Convention.
- Published
- 2021
24. Measurement of oxygen isotope ratios (18O/16O) of aqueous O2in small samples by gas chromatography/isotope ratio mass spectrometry
- Author
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Jakov Bolotin, Hans-Peter E. Kohler, Thomas B. Hofstetter, Roland A. Werner, Matthias S. Brennwald, and Sarah G. Pati
- Subjects
Aqueous solution ,Chromatography ,Chemistry ,Organic Chemistry ,Extraction (chemistry) ,Fractionation ,010501 environmental sciences ,010402 general chemistry ,Molecular sieve ,01 natural sciences ,Isotopes of oxygen ,0104 chemical sciences ,Analytical Chemistry ,Kinetic isotope effect ,Gas chromatography ,Isotope-ratio mass spectrometry ,Spectroscopy ,0105 earth and related environmental sciences - Abstract
Rationale Oxygen isotope fractionation of molecular O2 is an important process for the study of aerobic metabolism, photosynthesis, and formation of reactive oxygen species. The latter is of particular interest for investigating the mechanism of enzyme-catalyzed reactions, such as the oxygenation of organic pollutants, which is an important detoxification mechanism. Methods We developed a simple method to measure the δ18O values of dissolved O2 in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N2 headspace, the dissolved O2 partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph. Results In aqueous samples of 10 mL and in diluted air samples, we quantified the δ18O values at O2 concentrations of 16 μM and 86 μM, respectively. The chromatographic separation of O2 and N2 with a molecular sieve column made it possible to use N2 as the headspace gas for the extraction of dissolved O2 from water. We were therefore able to apply a rigorous δ18O blank correction for the quantification of 18O/16O ratios in 20 nmol of injected O2. Conclusions The successful quantification of 18O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O2 illustrates how the proposed method can be applied for studying enzymatic O2 activation mechanisms in a variety of (bio)chemical processes. Copyright © 2016 John Wiley & Sons, Ltd.
- Published
- 2016
25. Systematic Exploration of Biotransformation Reactions of Amine- Containing Micropollutants in Activated Sludge
- Author
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Kathrin Fenner, Ulf Meier, Emma L. Schymanski, Samuel Derrer, Hans-Peter E. Kohler, Daniel Rentsch, Rebekka Gulde, and Damian E. Helbling
- Subjects
0301 basic medicine ,Primary (chemistry) ,Tertiary amine ,Sewage ,Chemistry ,General Chemistry ,010501 environmental sciences ,Wastewater ,01 natural sciences ,6. Clean water ,03 medical and health sciences ,030104 developmental biology ,Activated sludge ,Biodegradation, Environmental ,Biotransformation ,13. Climate action ,Environmental Chemistry ,Organic chemistry ,Amine gas treating ,Amines ,Activated sludge system ,Water Pollutants, Chemical ,0105 earth and related environmental sciences - Abstract
The main removal process for polar organic micropollutants during activated sludge treatment is biotransformation, which often leads to the formation of stable transformation products (TPs). Because the analysis of TPs is challenging, the use of pathway prediction systems can help by generating a list of suspected TPs. To complete and refine pathway prediction, comprehensive biotransformation studies for compounds exhibiting pertinent functional groups under environmentally relevant conditions are needed. Because many polar organic micropollutants present in wastewater contain one or several amine functional groups, we systematically explored amine biotransformation by conducting experiments with 19 compounds that contained 25 structurally diverse primary, secondary, and tertiary amine moieties. The identification of 144 TP candidates and the structure elucidation of 101 of these resulted in a comprehensive view on initial amine biotransformation reactions. The reactions with the highest relevance were N-oxidation, N-dealkylation, N-acetylation, and N-succinylation. Whereas many of the observed reactions were similar to those known for the mammalian metabolism of amine-containing xenobiotics, some N-acylation reactions were not previously described. In general, different reactions at the amine functional group occurred in parallel. Finally, recommendations on how these findings can be implemented to improve microbial pathway prediction of amine-containing micropollutants are given.
- Published
- 2016
26. Enzyme Kinetics of Organic Contaminant Oxygenations
- Author
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Hans-Peter E. Kohler, Charlotte E. Bopp, and Thomas B. Hofstetter
- Subjects
Oxygenase ,Substrate (chemistry) ,General Medicine ,General Chemistry ,Metabolism ,Oxidative phosphorylation ,Biodegradation ,Hydroxylation ,biodegradation ,catalytic cycles ,Dioxygenases ,Oxygen ,lcsh:Chemistry ,Kinetics ,chemistry.chemical_compound ,lcsh:QD1-999 ,chemistry ,Biotransformation ,Oxygenases ,Biophysics ,Enzyme kinetics ,Oxidation-Reduction ,non-heme fe ii oxygenases - Abstract
Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O2, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O2 uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O2 activation to substrate hydroxylation, depending on the extent of O–O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O2 uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively., Chimia, 74 (3), ISSN:0009-4293
- Published
- 2020
27. Catabolism of the groundwater micropollutant 2,6-dichlorobenzamide beyond 2,6-dichlorobenzoate is plasmid encoded in Aminobacter sp MSH1
- Author
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Baptiste Leroy, Rob Lavigne, Ruddy Wattiez, Benjamin Horemans, Dirk Springael, Raffaella Tassoni, Bart Raes, Hans-Peter E. Kohler, Vera van Noort, Cédric Lood, and Jeroen T’Syen
- Subjects
0301 basic medicine ,GENES ,030106 microbiology ,2,6-Dichlorobenzamide ,Applied Microbiology and Biotechnology ,Amidase ,Amidohydrolases ,Dioxygenases ,03 medical and health sciences ,Plasmid ,GAMMA-HEXACHLOROCYCLOHEXANE ,3-CHLOROBENZOIC ACID ,Bacterial Proteins ,Dioxygenase ,NUCLEOTIDE-SEQUENCE ,COMAMONAS-TESTOSTERONI T-2 ,MOLECULAR CHARACTERIZATION ,Insertion sequence ,Gene ,Groundwater ,Drinking water treatment ,Aminobacter ,Science & Technology ,biology ,Chemistry ,Catabolism ,MICROBIAL GENOMES ,Plasmid encoded ,AEROBIC DEGRADATION ,General Medicine ,Phyllobacteriaceae ,HERBICIDE DICHLOBENIL ,Integrase ,Chlorobenzoates ,Metabolic pathway ,030104 developmental biology ,Biodegradation, Environmental ,METABOLITE 2,6-DICHLOROBENZAMIDE ,Biochemistry ,Biotechnology & Applied Microbiology ,Benzamides ,Catabolic pathway ,biology.protein ,Life Sciences & Biomedicine ,Water Pollutants, Chemical ,Biotechnology ,Plasmids - Abstract
Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as sole source of carbon and energy. In the first step, MSH1 converts BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) by means of the BbdA amidase encoded on the IncP-1β plasmid pBAM1. Information about the genes and degradation steps involved in 2,6-DCBA metabolism in MSH1 or any other organism is currently lacking. Here, we show that the genes for 2,6-DCBA degradation in strain MSH1 reside on a second catabolic plasmid in MSH1, designated as pBAM2. The complete sequence of pBAM2 was determined revealing that it is a 53.9 kb repABC family plasmid. The 2,6-DCBA catabolic genes on pBAM2 are organized in two main clusters bordered by IS elements and integrase genes and encode putative functions like Rieske mono-/dioxygenase, meta-cleavage dioxygenase, and reductive dehalogenases. The putative mono-oxygenase encoded by the bbdD gene was shown to convert 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoate (3-OH-2,6-DCBA). 3-OH-DCBA was degraded by wild-type MSH1 and not by a pBAM2-free MSH1 variant indicating that it is a likely intermediate in the pBAM2-encoded DCBA catabolic pathway. Based on the activity of BbdD and the putative functions of the other catabolic genes on pBAM2, a metabolic pathway for BAM/2,6-DCBA in strain MSH1 was suggested. ispartof: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY vol:102 issue:18 pages:7963-7979 ispartof: location:Germany status: published
- Published
- 2018
28. Biodegradation of synthetic polymers in soils: Tracking carbon into CO2 and microbial biomass
- Author
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Taylor F. Nelson, Hans-Peter E. Kohler, Michael Wagner, Rebekka Baumgartner, Dagmar Woebken, Kristopher McNeill, Michael T. Zumstein, Michael Sander, and Arno Schintlmeister
- Subjects
Polymers ,Polyesters ,Environmental Studies ,Biomass ,chemistry.chemical_element ,Spectrometry, Mass, Secondary Ion ,02 engineering and technology ,010501 environmental sciences ,01 natural sciences ,complex mixtures ,Soil Microbiology ,Research Articles ,0105 earth and related environmental sciences ,2. Zero hunger ,chemistry.chemical_classification ,Carbon Isotopes ,Multidisciplinary ,Fungi ,SciAdv r-articles ,Agriculture ,Polymer ,Lipase ,Biodegradation ,Carbon Dioxide ,021001 nanoscience & nanotechnology ,Pulp and paper industry ,Biodegradable polymer ,Carbon ,Polyester ,Biodegradation, Environmental ,chemistry ,13. Climate action ,Soil water ,Environmental science ,0210 nano-technology ,Soil microbiology ,Research Article - Abstract
Stable isotope labeling of agricultural polyesters enables demonstration of their microbial utilization in soils., Plastic materials are widely used in agricultural applications to achieve food security for the growing world population. The use of biodegradable instead of nonbiodegradable polymers in single-use agricultural applications, including plastic mulching, promises to reduce plastic accumulation in the environment. We present a novel approach that allows tracking of carbon from biodegradable polymers into CO2 and microbial biomass. The approach is based on 13C-labeled polymers and on isotope-specific analytical methods, including nanoscale secondary ion mass spectrometry (NanoSIMS). Our results unequivocally demonstrate the biodegradability of poly(butylene adipate-co-terephthalate) (PBAT), an important polyester used in agriculture, in soil. Carbon from each monomer unit of PBAT was used by soil microorganisms, including filamentous fungi, to gain energy and to form biomass. This work advances both our conceptual understanding of polymer biodegradation and the methodological capabilities to assess this process in natural and engineered environments.
- Published
- 2018
29. Ion Trapping of Amines in Protozoa: A Novel Removal Mechanism for Micropollutants in Activated Sludge
- Author
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Sabine Anliker, Kathrin Fenner, Hans-Peter E. Kohler, and Rebekka Gulde
- Subjects
0301 basic medicine ,010501 environmental sciences ,Wastewater ,Photochemistry ,01 natural sciences ,Ion trapping ,Waste Disposal, Fluid ,Cell membrane ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Environmental Chemistry ,Amines ,0105 earth and related environmental sciences ,biology ,Sewage ,Chemistry ,Vesicle ,Acridine orange ,General Chemistry ,biology.organism_classification ,Fluorescence ,Kinetics ,030104 developmental biology ,medicine.anatomical_structure ,Activated sludge ,Environmental chemistry ,Protozoa ,Amine gas treating ,Water Pollutants, Chemical - Abstract
To optimize removal of organic micropollutants from the water cycle, understanding the processes during activated sludge treatment is essential. In this study, we hypothesize that aliphatic amines, which are highly abundant among organic micropollutants, are partly removed from the water phase in activated sludge through ion trapping in protozoa. In ion trapping, which has been extensively investigated in medical research, the neutral species of amine-containing compounds diffuse through the cell membrane and further into acidic vesicles present in eukaryotic cells such as protozoa. There they become trapped because diffusion of the positively charged species formed in the acidic vesicles is strongly hindered. We tested our hypothesis with two experiments. First, we studied the distribution of the fluorescent amine acridine orange in activated sludge by confocal fluorescence imaging. We observed intense fluorescence in distinct compartments of the protozoa, but not in the bacterial biomass. Second, we investigated the distribution of 12 amine-containing and eight control micropollutants in both regular activated sludge and sludge where the protozoa had been inactivated. In contrast to most control compounds, the amine-containing micropollutants displayed a distinctly different behavior in the noninhibited sludge compared to the inhibited one: (i) more removal from the liquid phase; (ii) deviation from first-order kinetics for the removal from the liquid phase; and (iii) higher amounts in the solid phase. These results provide strong evidence that ion trapping in protozoa occurs and that it is an important removal mechanism for amine-containing micropollutants in batch experiments with activated sludge that has so far gone unnoticed. We expect that our findings will trigger further investigations on the importance of this process in full-scale wastewater treatment systems, including its relevance for accumulation of ammonium.
- Published
- 2017
30. Isolation of the (+)-Pinoresinol-Mineralizing Pseudomonas sp. Strain SG-MS2 and Elucidation of Its Catabolic Pathway
- Author
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Gunjan Pandey, Daniel Rentsch, Andrew C. Warden, David M. Cahill, Michael J. Lacey, Hans-Peter E. Kohler, Madhura Shettigar, Sahil Balotra, and John G. Oakeshott
- Subjects
0301 basic medicine ,Stereochemistry ,030106 microbiology ,Cometabolism ,Applied Microbiology and Biotechnology ,Lignin ,Lignans ,Hydroxylation ,03 medical and health sciences ,chemistry.chemical_compound ,Calcification, Physiologic ,4-Butyrolactone ,Pseudomonas ,Vanillic acid ,Humans ,Furans ,Lignan ,Minerals ,Ecology ,Quinone methide ,Gastrointestinal Microbiome ,030104 developmental biology ,chemistry ,Pinoresinol ,Benzaldehydes ,Biodegradation ,Metabolic Networks and Pathways ,Food Science ,Biotechnology ,Coniferyl alcohol - Abstract
Pinoresinol is a dimer of two β-β′-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several β-β-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2. IMPORTANCE The oxidative catabolism of (+)-pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.
- Published
- 2017
31. Stereochemistry of enzymatic transformations of (+)β- and (−)β-HBCD with LinA2 – A HCH-degrading bacterial enzyme of Sphingobium indicum B90A
- Author
-
Birgit Geueke, Peter Lienemann, Heidi Moor, Thomas Fleischmann, W. Bernd Schweizer, Simon A. Wyss, Hans-Peter E. Kohler, and Norbert V. Heeb
- Subjects
Environmental Engineering ,Stereochemistry ,Health, Toxicology and Mutagenesis ,Metabolite ,Molecular Conformation ,Stereoisomerism ,Catalysis ,chemistry.chemical_compound ,Bacterial Proteins ,Environmental Chemistry ,biology ,Public Health, Environmental and Occupational Health ,Cationic polymerization ,General Medicine ,General Chemistry ,biology.organism_classification ,Pollution ,Hydrocarbons, Brominated ,Sphingomonadaceae ,chemistry ,Docking (molecular) ,Enantiomer ,Hexachlorocyclohexane ,Sphingobium indicum - Abstract
LinA2, a bacterial enzyme expressed in various Sphingomonadaceae, catalyzes the elimination of HCl from hexachlorocyclohexanes (HCHs) and, as discussed here, the release of HBr from certain hexabromocyclododecanes (HBCDs). Both classes of compounds are persistent organic pollutants now regulated under the Stockholm Convention. LinA2 selectively catalyzes the transformation of β-HBCDs; other stereoisomers like α-, γ-, and δ-HBCDs are not converted. The transformation of (-)β-HBCD is considerably faster than that of its enantiomer. Here, we present the XRD crystal structure of 1E,5S,6S,9R,10S-pentabromocyclododecene (PBCDE) and demonstrate that its enantiomer with the 1E,5R,6R,9S,10R-configuration is the only metabolite formed during LinA2-catalyzed dehydrobromination of (-)β-HBCD. Formation of this product can be rationalized by HBr elimination at C5 and C6. A reasonable enzyme-substrate complex with the catalytic dyad His-73 and Asp-25 approaching the hydrogen at C6 and a cationic pocket of Lys-20, Try-42 and Arg-129 binding the leaving bromine at C5 was found from in silico docking experiments. A second PBCDE of yet unknown configuration was obtained from (+)β-HBCD. We predicted its stereochemistry to be 1E,5S,6S,9S,10R-PBCDE from docking experiments. The enzyme-substrate complex obtained from LinA2 and an activated conformation of (+)β-HBCD allows the HBr elimination at C9 and C10 leading to the predicted product. Both modeled enzyme-substrate complexes are in line with 1,2-diaxial HBr eliminations. In conclusion, LinA2, a bacterial enzyme of the HCH-degrading strain Sphingobium indicum B90A was able to stereoselectively convert β-HBCDs. Configurations of both PBCDE metabolites were predicted by molecular docking experiments and confirmed in one case by XRD data.
- Published
- 2015
32. Association of Biodiversity with the Rates of Micropollutant Biotransformations among Full-Scale Wastewater Treatment Plant Communities
- Author
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Damian E. Helbling, Tae Kwon Lee, Martin Ackermann, Joonhong Park, Hans-Peter E. Kohler, David R. Johnson, and Kathrin Fenner
- Subjects
DNA, Bacterial ,Bacteria ,Ecology ,Water pollutants ,Molecular Sequence Data ,Biodiversity ,Plant community ,Sequence Analysis, DNA ,Wastewater ,Biology ,Applied Microbiology and Biotechnology ,Water Purification ,Functional biodiversity ,Ecosystem services ,Biodegradation ,Water Pollutants ,Sewage treatment ,Ecosystem ,Biotransformation ,Food Science ,Biotechnology - Abstract
Biodiversities can differ substantially among different wastewater treatment plant (WWTP) communities. Whether differences in biodiversity translate into differences in the provision of particular ecosystem services, however, is under active debate. Theoretical considerations predict that WWTP communities with more biodiversity are more likely to contain strains that have positive effects on the rates of particular ecosystem functions, thus resulting in positive associations between those two variables. However, if WWTP communities were sufficiently biodiverse to nearly saturate the set of possible positive effects, then positive associations would not occur between biodiversity and the rates of particular ecosystem functions. To test these expectations, we measured the taxonomic biodiversity, functional biodiversity, and rates of 10 different micropollutant biotransformations for 10 full-scale WWTP communities. We have demonstrated that biodiversity is positively associated with the rates of specific, but not all, micropollutant biotransformations. Thus, one cannot assume whether or how biodiversity will associate with the rate of any particular micropollutant biotransformation. We have further demonstrated that the strongest positive association is between biodiversity and the collective rate of multiple micropollutant biotransformations. Thus, more biodiversity is likely required to maximize the collective rates of multiple micropollutant biotransformations than is required to maximize the rate of any individual micropollutant biotransformation. We finally provide evidence that the positive associations are stronger for rare micropollutant biotransformations than for common micropollutant biotransformations. Together, our results are consistent with the hypothesis that differences in biodiversity can indeed translate into differences in the provision of particular ecosystem services by full-scale WWTP communities.
- Published
- 2015
33. Characterization of Substrate, Cosubstrate, and Product Isotope Effects Associated With Enzymatic Oxygenations of Organic Compounds Based on Compound-Specific Isotope Analysis
- Author
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Sarah G, Pati, Hans-Peter E, Kohler, and Thomas B, Hofstetter
- Subjects
Oxygen ,Kinetics ,Biodegradation, Environmental ,Isotopes ,Biocatalysis ,Environmental Pollutants ,Nitrobenzenes ,Dioxygenases ,Enzyme Assays ,Toluene - Abstract
Enzymatic oxygenations are among the most important biodegradation and detoxification reactions of organic pollutants. In the environment, however, such natural attenuation processes are extremely difficult to monitor. Changes of stable isotope ratios of aromatic pollutants at natural isotopic abundances serve as proxies for isotope effects associated with oxygenation reactions. Such isotope fractionations offer new avenues for revealing the pathway and extent of pollutant transformation and provide new insights into the mechanisms of catalysis by Rieske non-heme ferrous iron oxygenases. Based on compound-specific C, H, N, and O isotope analysis, we present a comprehensive methodology with which isotope effects can be derived from the isotope fractionation measured in substrates, the cosubstrate O
- Published
- 2017
34. FMNH
- Author
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Benjamin, Ricken, Boris A, Kolvenbach, Christian, Bergesch, Dirk, Benndorf, Kevin, Kroll, Hynek, Strnad, Čestmír, Vlček, Ricardo, Adaixo, Frederik, Hammes, Patrick, Shahgaldian, Andreas, Schäffer, Hans-Peter E, Kohler, and Philippe F-X, Corvini
- Subjects
Actinobacteria ,Sulfonamides ,Biodegradation, Environmental ,Flavin Mononucleotide ,Genes, Bacterial ,Multigene Family ,Carbon Radioisotopes ,Phylogeny ,Article ,Anti-Bacterial Agents ,Hydroquinones ,Mixed Function Oxygenases - Abstract
We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.
- Published
- 2017
35. Enzymatic Hydrolysis of Polyester Thin Films at the Nanoscale: Effects of Polyester Structure and Enzyme Active-Site Accessibility
- Author
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Daniela Rechsteiner, Hans-Peter E. Kohler, Kristopher McNeill, Veronika Perz, Nicolas Roduner, Georg M. Guebitz, Michael T. Zumstein, Michael Sander, and Doris Ribitsch
- Subjects
0301 basic medicine ,Cutinase ,Polymers ,Polyesters ,010501 environmental sciences ,01 natural sciences ,03 medical and health sciences ,Hydrolysis ,Enzymatic hydrolysis ,Catalytic Domain ,Environmental Chemistry ,Organic chemistry ,Lipase ,0105 earth and related environmental sciences ,chemistry.chemical_classification ,biology ,Chemistry ,General Chemistry ,Polymer ,Biodegradation ,Polyester ,030104 developmental biology ,Dicarboxylic acid ,Biodegradation, Environmental ,biology.protein - Abstract
Biodegradable polyesters have a large potential to replace persistent polymers in numerous applications and to thereby reduce the accumulation of plastics in the environment. Ester hydrolysis by extracellular carboxylesterases is considered the rate-limiting step in polyester biodegradation. In this work, we systematically investigated the effects of polyester and carboxylesterase structure on the hydrolysis of nanometer-thin polyester films using a quartz-crystal microbalance with dissipation monitoring. Hydrolyzability increased with increasing polyester-chain flexibility as evidenced from differences in the hydrolysis rates and extents of aliphatic polyesters varying in the length of their dicarboxylic acid unit and of poly(butylene adipate-co-terephthalate) (PBAT) polyesters varying in their terephthalate-to-adipate ratio by Rhizopus oryzae lipase and Fusarium solani cutinase. Nanoscale nonuniformities in the PBAT films affected enzymatic hydrolysis and were likely caused by domains with elevated terephthalate contents that impaired enzymatic hydrolysis. Yet, the cutinase completely hydrolyzed all PBAT films, including films with a terephthalate-to-adipate molar ratio of one, under environmentally relevant conditions (pH 6, 20 °C). A comparative analysis of the hydrolysis of two model polyesters by eight different carboxylesterases revealed increasing hydrolysis with increasing accessibility of the enzyme active site. Therefore, this work highlights the importance of both polyester and carboxylesterase structure to enzymatic polyester hydrolysis.
- Published
- 2017
36. Characterization of Substrate, Cosubstrate, and Product Isotope Effects Associated With Enzymatic Oxygenations of Organic Compounds Based on Compound-Specific Isotope Analysis
- Author
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Hans-Peter E. Kohler, Thomas B. Hofstetter, and Sarah G. Pati
- Subjects
Isotope ,Stable isotope ratio ,010401 analytical chemistry ,010501 environmental sciences ,01 natural sciences ,0104 chemical sciences ,Nitrobenzene ,Isotopic labeling ,chemistry.chemical_compound ,Isotope fractionation ,chemistry ,Kinetic isotope effect ,Organic chemistry ,Isotope-ratio mass spectrometry ,0105 earth and related environmental sciences ,Isotope analysis - Abstract
Enzymatic oxygenations are among the most important biodegradation and detoxification reactions of organic pollutants. In the environment, however, such natural attenuation processes are extremely difficult to monitor. Changes of stable isotope ratios of aromatic pollutants at natural isotopic abundances serve as proxies for isotope effects associated with oxygenation reactions. Such isotope fractionations offer new avenues for revealing the pathway and extent of pollutant transformation and provide new insights into the mechanisms of catalysis by Rieske non-heme ferrous iron oxygenases. Based on compound-specific C, H, N, and O isotope analysis, we present a comprehensive methodology with which isotope effects can be derived from the isotope fractionation measured in substrates, the cosubstrate O2, and organic oxygenation products. We use dioxygenation of nitrobenzene and 2-nitrotoluene by nitrobenzene dioxygenase as illustrative examples to introduce different mathematical procedures for deriving apparent substrate and product isotope effects. We present two experimental approaches to control reactant and product turnover for isotope fractionation analysis in experimental systems containing purified enzymes, E. coli clones, and pure strains of environmental microorganisms. Finally, we present instrumental procedures and sample treatment instructions for analysis of C, H, and N isotope analysis in organic compounds and O isotope analysis in aqueous O2 by gas and liquid chromatography coupled to isotope ratio mass spectrometry.
- Published
- 2017
37. Important amino acid residues of hexachlorocyclohexane dehydrochlorinases (LinA) for enantioselective transformation of hexachlorocyclohexane isomers
- Author
-
Ankit S. Macwan, Hans-Peter E. Kohler, Nidhi Shrivastava, and Ashwani Kumar
- Subjects
0301 basic medicine ,Environmental Engineering ,Chromatography, Gas ,Stereochemistry ,030106 microbiology ,Mutant ,Hexachlorocyclohexane ,HCH dehydrochlorinase LinA ,Enantioselectivity ,alpha-HCH enantiomers ,Lyases ,Bioengineering ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,α-HCH enantiomers ,Environmental Chemistry ,Amino Acids ,chemistry.chemical_classification ,Original Paper ,Dehydrochlorinases ,Enantioselective synthesis ,Biochemistry and Molecular Biology ,Stereoisomerism ,Pollution ,Amino acid ,Transformation (genetics) ,030104 developmental biology ,Enzyme ,Biodegradation, Environmental ,chemistry ,Biochemistry ,Mutation ,Mutant Proteins ,Enantiomer ,Biokemi och molekylärbiologi - Abstract
LinA-type1 and LinA-type2 are two well-characterized variants of the enzyme ‘hexachlorocyclohexane (HCH)-dehydrochlorinase’. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (–) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (–) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinA-type1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes. Electronic supplementary material The online version of this article (doi:10.1007/s10532-017-9786-9) contains supplementary material, which is available to authorized users.
- Published
- 2017
38. FMNH2-dependent monooxygenases initiate catabolism of sulfonamides in Microbacterium sp. strain BR1 subsisting on sulfonamide antibiotics
- Author
-
Boris A. Kolvenbach, Čestmír Vlček, Patrick Shahgaldian, Benjamin Ricken, Andreas Schäffer, Ricardo Adaixo, Christian Bergesch, Frederik Hammes, Hans-Peter E. Kohler, Hynek Strnad, Kevin Kroll, Dirk Benndorf, and Philippe F.-X. Corvini
- Subjects
0301 basic medicine ,Multidisciplinary ,biology ,Catabolism ,medicine.drug_class ,Chemistry ,030106 microbiology ,Antibiotics ,lcsh:R ,lcsh:Medicine ,Monooxygenase ,biology.organism_classification ,Relaxase ,3. Good health ,Microbiology ,03 medical and health sciences ,Biochemistry ,FMN reductase ,Gene cluster ,medicine ,ddc:000 ,lcsh:Q ,lcsh:Science ,Gene ,Bacteria - Abstract
We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.
- Published
- 2017
39. LinA2, a HCH-converting bacterial enzyme that dehydrohalogenates HBCDs
- Author
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Birgit Geueke, Thomas Fleischmann, Peter Lienemann, Hans-Peter E. Kohler, Norbert V. Heeb, and Simon A. Wyss
- Subjects
HCH-converting bacterial enzyme ,Environmental Engineering ,Halogenation ,Hydrolases ,Stereochemistry ,Health, Toxicology and Mutagenesis ,Stereoisomerism ,HBCD biotransformation ,Brominated flame retardants ,Environmental Chemistry ,Enantiomeric excess ,Biotransformation ,LinA2 metabolites ,Dehalogenase ,572: Biochemie ,Meso compound ,Chemistry ,Persistent organic pollutants ,Public Health, Environmental and Occupational Health ,Substrate (chemistry) ,Lindane ,Soil pollutants ,General Medicine ,General Chemistry ,Brominated hydrocarbons ,Pollution ,Hydrocarbons, Brominated ,Sphingomonadaceae ,Kinetics ,Biocatalysis ,Polystyrenes ,Hexachlorocyclohexane ,Haloalkane dehalogenase ,Sphingobium indicum - Abstract
Hexabromocyclododecanes (HBCDs) and hexachlorocyclohexanes (HCHs) are lipophilic, polyhalogenated hydrocarbons with comparable stereochemistry. Bacterial evolution in HCH-contaminated soils resulted in the development of several Spingomonadaceae which express a series of HCH-converting enzymes. We showed that LinB, a haloalkane dehalogenase from Sphingobium indicum B90A, also transforms various HBCDs besides HCHs. Here we present evidence that LinA2, another dehalogenase from S. indicum also converts certain HBCDs to pentabromocyclododecenes (PBCDEs). Racemic mixtures of α-, β-, γ-HBCDs, a mixture of them, and δ-HBCD, a meso form, were exposed to LinA2. Substantial conversion of (-)β-HBCD was observed, but all other stereoisomers were not transformed significantly. The enantiomeric excess (EE) of β-HBCDs increased up to 60% in 32 h, whereas EE values of α- and γ-HBCDs were not affected. Substrate conversion and product formation were described with second-order kinetic models. One major (P1β) and possibly two minor (P2β, P3β) metabolites were detected. Respective mass spectra showed the characteristic isotope pattern of PBCDEs, the HBr elimination products of HBCDs. Michaelis-Menten parameters KM=0.47 ± 0.07 μM and vmax=0.17 ± 0.01 μmoll(-1)h(-1) were deduced from exposure data with varying enzyme/substrate ratios. LinA2 is more substrate specific than LinB, the latter converted all tested HBCDs, LinA2 only one. The widespread HCH pollution favored the selection and evolution of bacteria converting these compounds. We found that LinA2 and LinB, two of these HCH-converting enzymes expressed in S. indicum B90A, also dehalogenate HBCDs to lower brominated compounds, indicating that structural similarities of both classes of compounds are recognized at the level of substrate-protein interactions.
- Published
- 2014
40. Small 13C/12C Fractionation Contrasts with Large Enantiomer Fractionation in Aerobic Biodegradation of Phenoxy Acids
- Author
-
Philip Weyrauch, Eva C. Magana Lopez, Martin Elsner, Sebastian R. Sørensen, Erkin Gözdereliler, Hans-Peter E. Kohler, Shiran Qiu, and Rainer U. Meckenstock
- Subjects
Carbon Isotopes ,Chromatography ,Bacteria ,biology ,Herbicides ,Stereoisomerism ,General Chemistry ,Fractionation ,Chemical Fractionation ,Biodegradation ,Phenoxyacetates ,MCPA ,Aerobiosis ,Enzyme assay ,chemistry.chemical_compound ,Biodegradation, Environmental ,Isotope fractionation ,chemistry ,Isotopes of carbon ,biology.protein ,Environmental Chemistry ,2,4-Dichlorophenoxyacetic Acid ,Enantiomer ,Enzyme Assays ,Isotope analysis - Abstract
Phenoxy acid herbicides are important groundwater contaminants. Stable isotope analysis and enantiomer analysis are well-recognized approaches for assessing in situ biodegradation in the field. In an aerobic degradation survey with six phenoxyacetic acid and three phenoxypropionic acid-degrading bacteria we measured (a) enantiomer-specific carbon isotope fractionation of MCPP ((R,S)-2-(4-chloro-2-methylphenoxy)-propionic acid), DCPP ((R,S)-2-(2,4-dichlorophenoxy)-propionic acid), and 4-CPP ((R,S)-2-(4-chlorophenoxy)-propionic acid); (b) compound-specific isotope fractionation of MCPA (4-chloro-2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid); and (c) enantiomer fractionation of MCPP, DCPP, and 4-CPP. Insignificant or very slight (ε = -1.3‰ to -2.0‰) carbon isotope fractionation was observed. Equally small values in an RdpA enzyme assay (εea = -1.0 ± 0.1‰) and even smaller fractionation in whole cell experiments of the host organism Sphingobium herbicidovorans MH (εwc = -0.3 ± 0.1‰) suggest that (i) enzyme-associated isotope effects were already small, yet (ii) further masked by active transport through the cell membrane. In contrast, enantiomer fractionation in MCPP, DCPP, and 4-CPP was pronounced, with enantioselectivities (ES) of -0.65 to -0.98 with Sphingomonas sp. PM2, -0.63 to -0.89 with Sphingobium herbicidovorans MH, and 0.74 to 0.97 with Delftia acidovorans MC1. To detect aerobic biodegradation of phenoxypropionic acids in the field, enantiomer fractionation seems, therefore, a stronger indicator than carbon isotope fractionation.
- Published
- 2014
41. Slow Biotransformation of Carbon Nanotubes by Horseradish Peroxidase
- Author
-
Andreas Schäffer, D. Xanat Flores-Cervantes, Juliane Hollender, Hanna Maes, and Hans-Peter E. Kohler
- Subjects
biology ,Nanotubes, Carbon ,Chemistry ,General Chemistry ,Carbon nanotube ,Spectrum Analysis, Raman ,Horseradish peroxidase ,law.invention ,Microscopy, Electron, Transmission ,Biotransformation ,Chemical engineering ,law ,Microscopy, Electron, Scanning ,biology.protein ,Scattering, Radiation ,Environmental Chemistry ,Organic chemistry ,Degradation (geology) ,Carbon Radioisotopes ,Oxidation-Reduction ,Horseradish Peroxidase - Abstract
Due to steady increase in use and mass production carbon nanotubes (CNTs) will inevitably end up in the environment. Because of their chemical nature CNTs are expected to be recalcitrant and biotransform only at very slow rates. Degradation of CNTs within days has recently been reported, but excluding one study, conclusions relied solely on qualitative results. We incubated 13 different types of CNTs and subjected them to enzymatic oxidation with horseradish peroxidase and concluded that the analytical methods commonly employed for studying degradation of CNTs did not have the sensitivity to unequivocally demonstrate degradation of these materials. To obtain unambiguous results with regard to the biotransformability of CNTs in the horseradish peroxidase system we incubated: (a) (14)C-labeled multiwalled CNTs, homologous to Baytubes CNTs; and (b) (13)C-depleted single-walled CNTs, used in previous studies. Our results show that (14)C-CO2 evolved linearly at a rate of about 0.02‰ per day, and at the end of the 30-day incubations the CO2 evolved amounted to about 0.5‰ of both initial substrates, the (14)C-labeled multiwalled and (13)C-depleted single-walled CNTs. These results clearly show that CNT material is oxidized in the horseradish peroxidase system but with half-lives of about 80 years and not a few days as has been reported before. Adequately addressing biotransformation rates of CNTs is key toward a better understanding of the fate of these materials in the environment.
- Published
- 2014
42. Kinetics and Yields of Pesticide Biodegradation at Low Substrate Concentrations and under Conditions Restricting Assimilable Organic Carbon
- Author
-
Thomas Egli, Damian E. Helbling, Hans-Peter E. Kohler, and Frederik Hammes
- Subjects
Chromatography ,Bacteria ,Ecology ,Environmental remediation ,Kinetics ,Substrate (chemistry) ,chemistry.chemical_element ,Biodegradation ,Bacterial growth ,Flow Cytometry ,Applied Microbiology and Biotechnology ,Carbon ,Mass Spectrometry ,Biotransformation ,chemistry ,Environmental chemistry ,Degradation (geology) ,Organic Chemicals ,Pesticides ,Chromatography, High Pressure Liquid ,Food Science ,Biotechnology - Abstract
The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter −1 . We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.
- Published
- 2014
43. Emerging chemicals and the evolution of biodegradation capacities and pathways in bacteria
- Author
-
Boris A Kolvenbach Damian E Helbling Hans-Peter E Kohler Philippe F-X Corvini
- Abstract
The number of new chemicals produced is increasing daily by the thousands and it is inevitable that many of these chemicals will reach the environment. Current research provides an understanding of how the evolution of promiscuous enzymes and the recruitment of enzymes available from the metagenome allows for the assembly of these pathways. Nevertheless physicochemical constraints including bioavailability bioaccessibility and the structural variations of similar chemicals limit the evolution of biodegradation pathways. Similarly physiological constraints related to kinetics and substrate utilization at low concentrations likewise limit chemical enzyme interactions and consequently evolution. Considering these new data the biodegradation decalogue still proves valid while at the same time the underlying mechanisms are better understood.
- Published
- 2014
44. Biotransformation of hexabromocyclododecanes with hexachlorocyclohexane-transforming Sphingobium chinhatense strain IP26
- Author
-
Peter Lienemann, Andreas Grubelnik, Birgit Geueke, Norbert V. Heeb, and Hans-Peter E. Kohler
- Subjects
0301 basic medicine ,Environmental Engineering ,Halogenation ,Health, Toxicology and Mutagenesis ,Hexachlorocyclohexane ,010501 environmental sciences ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Bioremediation ,Biotransformation ,Environmental Chemistry ,Enantiomeric excess ,0105 earth and related environmental sciences ,Flame Retardants ,biology ,Public Health, Environmental and Occupational Health ,Stereoisomerism ,General Medicine ,General Chemistry ,Pesticide ,biology.organism_classification ,Pollution ,Hydrocarbons, Brominated ,Sphingomonadaceae ,Transformation (genetics) ,030104 developmental biology ,Biodegradation, Environmental ,chemistry ,Environmental chemistry ,Environmental Pollutants ,Enantiomer ,Bacteria - Abstract
Bacterial evolution has resulted in the appearance of several Sphingomonadacea strains that gained the ability to metabolize hexachlorocyclohexanes (HCHs). HCHs have been widely used as pesticides but were banned under the Stockholm Convention on persistent organic pollutants (POPs) in 2009. Here we present evidence for bacterial transformation reactions of hexabromocyclododecanes (HBCDs), which are structurally related to HCHs. HBCDs were used as flame retardants. They are now also considered as POPs and their production and use is restricted since 2013. Racemic α-, β-, and γ-HBCDs and their mixture were exposed to Sphingobium chinhatense IP26 in resting cell assays in parallel to β-HCH. All HBCD stereoisomers were converted with (−)β-HBCD being the best and both α-HBCD enantiomers the poorest substrates. HBCD conversion rates were 27–430 times slower than that of β-HCH. Three generations of hydroxylated transformation products were observed, 7 pentabromocyclododecanol isomers (PeBCD-ols), 11 tetrabromocyclododecadiols (TeBCD-diols) and 3 tribromocyclododecatriols (TrBCD-triols). The conversion of (+)α-, (−)β- and (−)γ-HBCD was faster than those of their enantiomers. Therefore the respective enantiomeric excess increased to 3 ± 1%, 36 ± 1% and 6 ± 2% during 48 h of bacterial exposure. PeBCD-ols appeared first, followed by TeBCD-diols and TrBCD-triols indicating stepwise hydrolytic dehalogenation reactions. In conclusion, severe HCH pollution at geographically distinct dumpsites triggered bacterial evolution to express enzymes transforming such compounds. We used S. chinhatense IP26 bacteria to transform structurally related HBCDs, also regulated under the Stockholm Convention. Such bacteria might be useful for bioremediation but the toxicity of the numerous transformation products observed must be assessed in advance.
- Published
- 2016
45. NOM degradation during river infiltration: Effects of the climate variables temperature and discharge
- Author
-
Samuel Diem, Hans-Peter E. Kohler, Janet G. Hering, Matthias Rudolf von Rohr, Mario Schirmer, and Urs von Gunten
- Subjects
Environmental Engineering ,Denitrification ,Climate ,chemistry.chemical_element ,Manganese ,chemistry.chemical_compound ,Rivers ,Nitrate ,Mineral redox buffer ,Dissolved organic carbon ,Organic matter ,Organic Chemicals ,Groundwater ,Waste Management and Disposal ,Water Science and Technology ,Civil and Structural Engineering ,chemistry.chemical_classification ,Hydrology ,Nitrates ,Geography ,Ecological Modeling ,Temperature ,Water ,Pollution ,Oxygen ,Infiltration (hydrology) ,chemistry ,Environmental chemistry ,Water quality ,Oxidation-Reduction ,Filtration ,Switzerland - Abstract
Most peri-alpine shallow aquifers fed by rivers are oxic and the drinking water derived by riverbank filtration is generally of excellent quality. However, observations during past heat waves suggest that water quality may be affected by climate change due to effects on redox processes such as aerobic respiration, denitrification, reductive dissolution of manganese(III/IV)- and iron(III)(hydr)oxides that occur during river infiltration. To assess the dependence of these redox processes on the climate-related variables temperature and discharge, we performed periodic and targeted (summer and winter) field sampling campaigns at the Thur River, Switzerland, and laboratory column experiments simulating the field conditions. Typical summer and winter field conditions could be successfully simulated by the column experiments. Dissolved organic matter (DOM) was found not to be a major electron donor for aerobic respiration in summer and the DOM consumption did not reveal a significant correlation with temperature and discharge. It is hypothesized that under summer conditions, organic matter associated with the aquifer material (particulate organic matter, POM) is responsible for most of the consumption of dissolved oxygen (DO), which was the most important electron acceptor in both the field and the column system. For typical summer conditions at temperatures20 °C, complete depletion of DO was observed in the column system and in a piezometer located only a few metres from the river. Both in the field system and the column experiments, nitrate acted as a redox buffer preventing the release of manganese(II) and iron(II). For periodic field observations over five years, DO consumption showed a pronounced temperature dependence (correlation coefficient r = 0.74) and therefore a seasonal pattern, which seemed to be mostly explained by the temperature dependence of the calculated POM consumption (r = 0.7). The river discharge was found to be highly and positively correlated with DO consumption (r = 0.85), suggesting an enhanced POM input during flood events. This high correlation could only be observed for the low-temperature range (T15 °C). For temperatures15 °C, DO consumption was already high (almost complete) and the impact of discharge could not be resolved. Based on our results, we estimate the risk for similar river-infiltration systems to release manganese(II) and iron(II) to be low during future average summer conditions. However, long-lasting heat waves might lead to a consumption of the nitrate buffer, inducing a mobilization of manganese and iron.
- Published
- 2013
46. ipso-Hydroxylation and Subsequent Fragmentation: a Novel Microbial Strategy To Eliminate Sulfonamide Antibiotics
- Author
-
Benjamin Ricken, Danuta Cichocka, Boris A. Kolvenbach, Philippe F.-X. Corvini, Ludovico G. Tulli, Dominik Wyss, Hans-Peter E. Kohler, Martina Parisi, Markus Lenz, Jochen A. Müller, Paula M. Martínez-Lavanchy, Patrick Shahgaldian, B. Ricken, P. F. X. Corvini, D. Cichocka, M. Parisi, M. Lenz, D. Wy, P. M. Martinez-Lavanchy, J. A. Muller, P. Shahgaldian, L. G. Tulli, H.-P. E. Kohler, and B. A. Kolvenbach
- Subjects
Stereochemistry ,WASTEWATER TREATMENT ,Substituent ,Sulfadimethoxine ,waste-water treatment ,Sulfamethizole ,010501 environmental sciences ,Hydroxylation ,01 natural sciences ,Applied Microbiology and Biotechnology ,biodegradation ,03 medical and health sciences ,chemistry.chemical_compound ,Sulfadiazine ,Biotransformation ,Actinomycetales ,substitution ,medicine ,0105 earth and related environmental sciences ,degradation ,chemistry.chemical_classification ,Sulfonyl ,0303 health sciences ,Sulfonamides ,WIMEK ,Ecology ,030306 microbiology ,resistance genes ,NAD ,cytochrome-p450 ,sphingobium-xenophagum bayram ,3. Good health ,Sulfonamide ,Anti-Bacterial Agents ,bisphenol-a ,chemistry ,escherichia-coli ,Environmental Technology ,Environmental Pollutants ,Milieutechnologie ,metabolism ,Metabolic Networks and Pathways ,Food Science ,Biotechnology ,medicine.drug - Abstract
Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso -hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino- N -phenylbenzenesulfonamide, and N -(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso -hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para -position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.
- Published
- 2013
47. Metabolomics of hexachlorocyclohexane (HCH) transformation: ratio of LinA to LinB determines metabolic fate of HCH isomers
- Author
-
Birgit Geueke, Nidhi Garg, Thomas Fleischmann, Christof Holliger, Hans-Peter E. Kohler, Sneha Ghosh, and Rup Lal
- Subjects
chemistry.chemical_classification ,Hexachlorocyclohexane ,Microbial metabolism ,Metabolism ,Biology ,Microbiology ,Hydroxylation ,chemistry.chemical_compound ,Bioremediation ,Enzyme ,chemistry ,Biochemistry ,Lindane ,Ecology, Evolution, Behavior and Systematics ,Haloalkane dehalogenase - Abstract
Although the production and use of technical hexachlorocyclohexane (HCH) and lindane (the purified insecticidal isomer γ-HCH) are prohibited in most countries, residual concentrations still constitute an immense environmental burden. Many studies describe the mineralization of γ-HCH by bacterial strains under aerobic conditions. However, the metabolic fate of the other HCH isomers is not well known. In this study, we investigated the transformation of α-, β-, γ-, δ-, e-HCH, and a heptachlorocyclohexane isomer in the presence of varying ratios of the two enzymes that initiate γ-HCH degradation, a dehydrochlorinase (LinA) and a haloalkane dehalogenase (LinB). Each substrate yielded a unique metabolic profile that was strongly dependent on the enzyme ratio. Comparison of these results to those of in vivo experiments with different bacterial isolates showed that HCH transformation in the tested strains was highly optimized towards productive metabolism of γ-HCH and that under these conditions other HCH-isomers were metabolized to mixtures of dehydrochlorinated and hydroxylated side-products. In view of these results, bioremediation efforts need very careful planning and toxicities of accumulating metabolites need to be evaluated.
- Published
- 2012
48. Bacterialβ-Aminopeptidases: Structural Insights and Applications for Biocatalysis
- Author
-
Tobias Heck, Birgit Geueke, and Hans-Peter E. Kohler
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Molecular Sequence Data ,Bioengineering ,Aminopeptidases ,Biochemistry ,Amidohydrolases ,Hydrolysis ,Protein structure ,Nucleophile ,Hydrolase ,Amino Acid Sequence ,Amino Acids ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Bacteria ,Chemistry ,General Chemistry ,General Medicine ,Amino acid ,Enzyme ,Biocatalysis ,Molecular Medicine ,Peptides - Abstract
β-Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β-aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β(3) - and/or β(2) -amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β-peptide substrates, β-aminopeptidases have potential to be used as biocatalysts for β-peptide synthesis and for the resolution of enantiomerically pure β-amino acids from racemic substrate mixtures. β-Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N-terminus of the newly formed β-polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N-terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β-aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β-aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β-peptide conversion.
- Published
- 2012
49. Autoproteolytic and Catalytic Mechanisms for the β-Aminopeptidase BapA—A Member of the Ntn Hydrolase Family
- Author
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Peer R. E. Mittl, Hans-Peter E. Kohler, Birgit Geueke, Christophe Briand, Dieter Seebach, Tobias Heck, Markus G. Grütter, Tobias M. Merz, University of Zurich, and Grütter, Markus G
- Subjects
Models, Molecular ,Stereochemistry ,Sphingosinicella xenopeptidilytica ,Crystallography, X-Ray ,Glutamyl Aminopeptidase ,010402 general chemistry ,01 natural sciences ,Aminopeptidase ,Catalysis ,Amidohydrolases ,03 medical and health sciences ,1315 Structural Biology ,Structural Biology ,Hydrolase ,10019 Department of Biochemistry ,1312 Molecular Biology ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,Active site ,0104 chemical sciences ,Biochemistry ,Ntn hydrolase ,Proteolysis ,biology.protein ,570 Life sciences ,Substrate specificity ,Homotetramer - Abstract
SUMMARY The b-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal b-amino acid residues from peptides. We determined the crystal structures of the native (ab)4 heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Au , respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.
- Published
- 2012
- Full Text
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50. Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants
- Author
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Hans-Peter E. Kohler, Sarah G. Pati, Rebecca E. Parales, Anna Pabis, Thomas B. Hofstetter, and Piotr Paneth
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inorganic chemicals ,0301 basic medicine ,010501 environmental sciences ,01 natural sciences ,Dioxygenases ,Substrate Specificity ,Chemical kinetics ,Nitrobenzene ,03 medical and health sciences ,chemistry.chemical_compound ,Isotope fractionation ,Biotransformation ,Dioxygenase ,Kinetic isotope effect ,Environmental Chemistry ,Organic chemistry ,0105 earth and related environmental sciences ,Naphthalene ,Ekologi ,Carbon Isotopes ,Ecology ,Nitrogen Isotopes ,Chemistry ,Substrate (chemistry) ,General Chemistry ,Kinetics ,030104 developmental biology ,Biodegradation, Environmental - Abstract
Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent C-13- and H-2-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O-2 activation and aromatic C oxygenation. The contribution of enzymatic O-2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O-2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.
- Published
- 2016
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