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Isotope Effects of Enzymatic Dioxygenation of Nitrobenzene and 2‑Nitrotoluene by Nitrobenzene Dioxygenase
journal contribution
posted on 2014-09-16, 00:00 authored by Sarah
G. Pati, Hans-Peter E. Kohler, Jakov Bolotin, Rebecca E. Parales, Thomas B. HofstetterOxygenation
of aromatic rings is a frequent initial step in the
biodegradation of persistent contaminants, and the accompanying isotope
fractionation is increasingly used to assess the extent of transformation
in the environment. Here, we systematically investigated the dioxygenation
of two nitroaromatic compounds (nitrobenzene and 2-nitrotoluene) by
nitrobenzene dioxygenase (NBDO) to obtain insights into the factors
governing its C, H, and N isotope fractionation. Experiments were
carried out at different levels of biological complexity from whole
bacterial cells to pure enzyme. C, H, and N isotope enrichment factors
and kinetic isotope effects (KIEs) were derived from the compound-specific
isotope analysis of nitroarenes, whereas C isotope fractionation was
also quantified in the oxygenated reaction products. Dioxygenation
of nitrobenzene to catechol and 2-nitrotoluene to 3-methylcatechol
showed large C isotope enrichment factors, ϵC, of
−4.1 ± 0.2‰ and −2.5 ± 0.2‰,
respectively, and was observed consistently in the substrates and
dioxygenation products. ϵH- and ϵN-values were smaller, that is −5.7 ± 1.3‰ and
−1.0 ± 0.3‰, respectively. C isotope fractionation
was also identical in experiments with whole bacterial cells and pure
enzymes. The corresponding 13C-KIEs for the dioxygenation
of nitrobenzene and 2-nitrotoluene were 1.025 ± 0.001 and 1.018
± 0.001 and suggest a moderate substrate specificity. Our study
illustrates that dioxygenation of nitroaromatic contaminants exhibits
a large C isotope fractionation, which is not masked by substrate
transport and uptake processes and larger than dioxygenation of other
aromatic hydrocarbons.
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Nitrobenzene DioxygenaseOxygenationsubstrate transportnitroaromatic contaminants exhibitsdioxygenation productsKIEIsotope Effectsnitrobenzene dioxygenaseC isotope enrichment factorsnitroaromatic compoundsuptake processesisotope fractionationNBDOsubstrate specificityoxygenated reaction productsC isotope fractionationN isotope fractionationN isotope enrichment factorsϵ Hϵ Cisotope effectsEnzymatic Dioxygenation
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