Enzyme Kinetics of Different Types of Flavin-Dependent Monooxygenases Determine the Observable Contaminant Stable Isotope Fractionation

Abstract

The assessment of oxidative pollutant biotransformation by compound specific isotope analysis (CSIA) is often complicated by the variability of kinetic isotope effects associated with carbon oxygenation in enzymatic reactions. Here, we illustrate how information about the kinetics of oxidative biocatalysis by flavin-dependent monooxygenases (FMOs) enables one to assess if CSIA could be applied for tracking contaminant biodegradation. In "cautious" FMOs, which form reactive flavin (hydro)peroxide species after substrate binding, the monooxygenation of organic compounds is not rate-determining and consequently does not lead to substrate isotope fractionation. Conversely, "bold" FMOs generate hydroperoxides regardless of substrate availability, and substrate disappearance is thus subject to isotope fractionation trends, which are typical for hydroxylation reactions. Because monooxygenations of aromatic moieties are often initial steps of organic pollutant transformation, knowledge of the kinetics of FMOs and other oxidative enzymes can support decisions regarding the use of CSIA.

Structure and function of a flavin-dependent S-monooxygenase from garlic (Allium sativum)

“…The kinetic characterization is consistent with AsFMO being a member the class B flavin-dependent monooxygenase mechanism (52). Members of this class share specificity for NADPH, pro-R stereoselectivity, and stabilization of the C4a-hydroperoxyflavin, as demonstrated for AsFMO (29, [53][54][55].…”

Characterization of Substrate, Cosubstrate, and Product Isotope Effects Associated With Enzymatic Oxygenations of Organic Compounds Based on Compound-Specific Isotope Analysis

“…Enzymatic oxygenations are among the most important biodegradation reactions in the environment, and they also contribute to initial transformation of numerous organic pollutants through co-metabolic oxygenations. Despite its relevance, knowledge of enzymatic mechanisms of O 2 activation and oxygenation of organic pollutants are scarce (e.g., Wijker et al (2015)). It is therefore very challenging to assess whether such processes will happen unless the stable isotope fractionation of soil and water pollutants is understood in greater detail.…”

“…At present, we lack comprehensive knowledge of the mechanisms of mono-and dioxygenations of organic pollutants and the magnitude of isotope effects that are associated with the initial steps of the reactions. Moreover, the kinetic complexity of enzymatic O 2 activation and its consequences for the expression of isotope fractionation in the organic substrate is poorly understood (Wijker, Pati, Zeyer, & Hofstetter, 2015). The isotope fractionation observed during enzyme-catalyzed oxygenations of organic pollutants can vary substantially even for identical reactions because of multiple rate-limiting steps.…”

δ13C and δ37Cl Isotope Fractionation To Characterize Aerobic vs Anaerobic Degradation of Trichloroethylene

“…Interpretation of CSIA could become challenging due to several different reasons, all driven from the fact that the apparent isotope effect cumulates various different factors that may diverge the observable isotope effect from the intrinsic isotope effect [45][46][47] . These include membrane induced equilibrium isotope effect 48 , equilibrium isotope effects on enzyme binding 49,50 or following product branching 51 . In these cases distinction e.g.…”