TY - JOUR
T1 - Isotope effects of enzymatic dioxygenation of nitrobenzene and 2-nitrotoluene by nitrobenzene dioxygenase
AU - Pati, Sarah G.
AU - Kohler, Hans Peter E.
AU - Bolotin, Jakov
AU - Parales, Rebecca E.
AU - Hofstetter, Thomas B.
N1 - Publisher Copyright:
© 2014 American Chemical Society.
PY - 2014/9/16
Y1 - 2014/9/16
N2 - Oxygenation 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.
AB - Oxygenation 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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U2 - 10.1021/es5028844
DO - 10.1021/es5028844
M3 - Article
C2 - 25101486
AN - SCOPUS:84924778002
SN - 0013-936X
VL - 48
SP - 10750
EP - 10759
JO - Environmental Science and Technology
JF - Environmental Science and Technology
IS - 18
ER -