The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.
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http://dx.doi.org/10.1111/1462-2920.16187 | DOI Listing |
Biotechnol Bioeng
October 2024
Department of Chemistry, Ball State University, Muncie, Indiana, USA.
Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high-value compounds, due to their capacity to perform the cis-dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis-dihydroxylation of benzoates.
View Article and Find Full Text PDFEnviron Sci Technol
March 2023
Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, Lodz 90-924, Poland.
Compound-specific isotope analysis (CSIA) for natural isotope ratios has been recognized as a promising tool to elucidate biodegradation pathways of organic pollutants by microbial enzymes by relating reported kinetic isotope effects (KIEs) to apparent KIEs (AKIEs) derived from bulk isotope fractionations (ε). However, for many environmental reactions, neither are the reference KIE ranges sufficiently narrow nor are the mechanisms elucidated to the point that rate-determining steps have been identified unequivocally. In this work, besides providing reference KIEs and rationalizing AKIEs, good relationships have been explained by DFT computations for diverse biodegradation pathways with known enzymatic models between the theoretical isotope fractionations (ε) from intrinsic KIEs on the rate-determining steps and the observed ε.
View Article and Find Full Text PDFJ Biotechnol
March 2023
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Electronic address:
Glycerol dehydrogenase (GldA) from Escherichia coli BW25113, naturally catalyzes the oxidation of glycerol to dihydroxyacetone. It is known that GldA exhibits promiscuity towards short-chain C-C alcohols. However, there are no reports regarding the substrate scope of GldA towards larger substrates.
View Article and Find Full Text PDFEnviron Microbiol
November 2022
Department of Biochemistry, Molecular Biology and Biophysics and Biotechnology Institute, University of Minnesota, Twin Cities, Minnesota, USA.
The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons.
View Article and Find Full Text PDFMolecules
June 2022
Department of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON L2S 3A1, Canada.
A 15-step chemoenzymatic total synthesis of C-1 methoxycarbonyl narciclasine () was accomplished. The synthesis began with the toluene dioxygenase-mediated dihydroxylation of -dibromobenzene to provide the corresponding -dihydrodiol () as a single enantiomer. Further key steps included a nitroso Diels-Alder reaction and an intramolecular Heck cyclization.
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