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18. Elucidating the Role of O2 Uncoupling in the Oxidative Biodegradation of Organic Contaminants by Rieske Non-heme Iron Dioxygenases

Author

Bopp, Charlotte E., Bernet Nora M., Kohler, Hans-Peter E., and Hofstetter, Thomas B.

Subjects
O2 uncoupling, non-heme ferrous iron oxygenases, Rieske oxygenases, biocatalysis, O2 activation, kinetic isotope effect, biodegradation
Abstract

Oxygenations of aromatic soil and water contaminants with molecular O2catalyzed by Rieske dioxygenases are frequent initial steps of biodegradation in natural and engineered environments. Many of these non-heme ferrous iron enzymes are known to be involved in contaminant metabolism, but the understanding of enzyme-substrate interactions that lead to successful biodegradation is still elusive. Here, we studied the mechanisms of O2activation and substrate hydroxylation of two nitroarene dioxygenases to evaluate enzyme- and substrate-specific factors that determine the efficiency of oxygenated product formation. Experiments in enzyme assays of 2-nitrotoluene dioxygenase (2NTDO) and nitrobenzene dioxygenase (NBDO) with methyl-, fluoro-, chloro-, and hydroxy-substituted nitroaromatic substrates reveal that typically 20-100% of the enzyme's activity involves unproductive paths of O2activation with generation of reactive oxygen species through so-called O2uncoupling. The 18O and 13C kinetic isotope effects of O2activation and nitroaromatic substrate hydroxylation, respectively, suggest that O2uncoupling occurs after generation of FeIII-(hydro)peroxo species in the catalytic cycle. While 2NTDO hydroxylates ortho-substituted nitroaromatic substrates more efficiently, NBDO favors meta-substituted, presumably due to distinct active site residues of the two enzymes. Our data implies, however, that the O2uncoupling and hydroxylation activity cannot be assessed from simple structure-reactivity relationships. By quantifying O2uncoupling by Rieske dioxygenases, our work provides a mechanistic link between contaminant biodegradation, the generation of reactive oxygen species, and possible adaptation strategies of microorganisms to the exposure of new contaminants., ACS Environmental Au, 2 (5)

Published
2022

23. Substrate-Specific Coupling of O2 Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases

Author

Pati, Sarah G., Bopp, Charlotte E., Kohler, Hans-Peter E., and Hofstetter, Thomas B.

Subjects
O2 uncoupling, isotope effects, nitrobenzene dioxygenase, non-heme ferrous iron oxygenases, biocatalysis, xenobiotics
Abstract

Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme FeII center, elementary steps leading to O2 activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O2 activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O2 uncoupling for a series of substituted nitroaromatic compounds with 18O and 13C kinetic isotope effects of dissolved O2 and aromatic substrates, respectively, we show that O2 uncoupling occurs after the rate-limiting formation of FeIII-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O2 uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O2 uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O2 activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases., ACS Catalysis, 12 (11), ISSN:2155-5435

Published
2022

34. Haloalkane dehalogenase LinB is responsible for [beta]- and [delta]-hexachlorocyclohexane transformation in 'Sphingobium indicum' B90A

Author

Sharma, Poonam, Raina, Vishakha, Kumari, Rekha, Malhotra, Shweta, Dogra, Charu, Kumari, Hansi, Kohler, Hans-Peter E., Buser, Hans-Rudolf, Holliger, Christof, and Lal Rup

Subjects
Bacterial transformation -- Research, Bacterial proteins -- Research, Chlorination -- Analysis, Biological sciences
Abstract

The potential of 'Sphingobium indicum' (S.indicum) B90A for transforming [beta]- and [delta]- hexachlorocyclohexane (HCH) is reconfirmed. It is demonstrated that LinB is responsible for the transformation of [beta]-HCH to pentachlorocyclohexanol and tetrachlorocyclohexanediol in 'S. indicum' B90A and that LinB also acts on delta-HCH, analogously forming the respective pentachlorocyclohexanol and tetrachlorocyclohexanediol intermediates.

Published
2006

35. Enantioselective transformation of alpha-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A

Author

Suar, Mrutyunjay, Hauser, Andrea, Poiger, Thomas, Buser, Hans-Rudolf, Muller, Markus D., Dogra, Charu, Raina, Vishaka, Holliger, Christof, van der Meer, Jan Roelof, Lal, Rup, and Kohler, Hans-Peter E.

Subjects
Lindane -- Chemical properties, Bacterial proteins -- Chemical properties, Escherichia coli -- Research, Soil microbiology -- Research, Biological sciences
Abstract

The enantioselective transformation of alpha-hexachlorocyclohexane (HCH) by LinA1 and LinA2 variants expressed as S-glutathione transferase (GST) fusion proteins in Escherichia coli BL21 is reported. Enantioselective degradation might be one of the key processes that determine enantiomer composition, especially when strains that contain only one of the linA genes, such as Sphingmonas paucimobilis UT26, prevail.

Published
2005

36. A novel [beta]-peptidyl aminopeptidase (BapA) from strain 3-2W4 cleaves peptide bonds of synthetic [beta]-tri- and [beta]-dipeptides

Author

Geueke, Birgit, Namoto, Kenji, Seebach, Dieter, and Kohler, Hans-Peter E.

Subjects
Aminopeptidases -- Genetic aspects, Peptides -- Genetic aspects, Biological sciences
Abstract

A novel bacterial strain that was capable of growing on the [beta]-tripeptide H-[beta]hVal-[beta]hAla-[beta]hLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the [beta]-tripeptide and the [beta]-dipeptide H-[beta]hAla-[beta]hLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated [beta]-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The [beta]-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a [beta]-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily.

Published
2005

37. Differential degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram

Author

Gabriel, Frederic L. P., Giger, Walter, Guenther, Klaus, and Kohler, Hans-Peter E.

Subjects
Biodegradation -- Research, Phenols -- Research, Biological sciences
Abstract

The isolation of an Sphingomonas xenophaga Bayram strain that efficiently degrades 4-(1-ethyl-1,4-dimethylpentyl)phenol is reported. The degradation experiments with five different 4-nonylphenol isomers have indicated that degradability is strongly dependent on the structure of the alkyl substituent.

Published
2005

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