Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase

Abstract

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP ؉ analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this "double-engineering" approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP ؉ in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogenbonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.Flavin-containing monooxygenases (FMOs) 3 form a large family of enzymes present almost ubiquitously among living organisms (1, 2, 3). They are involved in diverse biological processes such as the biosynthesis of various natural products (4) and catabolism of xenobiotics. Humans have five different isozymes that are coded by different genes and represent a key enzymatic system in drug metabolism, whose relevance almost equals that of cytochrome P450s. Among known FMO substrates, there are metabolites directly or indirectly deriving from food digestion such as trimethylamine, drugs such as tamoxifen, and toxic molecules such as nicotine. Mutations in the gene for the isozyme 3 of human FMO (FMO3), which is most abundant in the liver, are directly responsible for trimethylaminuria, also known as fish odor syndrome (TMAU). This genetic disease causes the accumulation of trimethylamine so that the body of the patients tend to have an unpleasant smell (5, 6).Irrespectively of their biological context, FMOs always catalyze the same reaction: the oxygenation of a soft nucleophile using molecular oxygen as oxygen source and NADPH as electron donor (7) (Fig. 1A). At the heart of this very complex reaction is the stabilization of the flavin-hydroperoxide adduct res...

Hydrogen Peroxide Elimination from C4a-hydroperoxyflavin in a Flavoprotein Oxidase Occurs through a Single Proton Transfer from Flavin N5 to a Peroxide Leaving Group

“…The model in Fig. 8 is also supported by the recent investigation of a flavin-containing monooxygenase showing that an N78S mutant, in which the site near the C4a/N5 locus is widened and in which the H-bonding interaction between the flavin N5 and NADP ϩ is altered, voids its ability to form a C4a-hydroperoxyflavin intermediate (41).…”

Human flavin-containing monooxygenase 3: Structural mapping of gene polymorphisms and insights into molecular basis of drug binding

“…2C). Since oxidized NADP + remains tightly bound and is involved in the stabilisation of the C4a-hydroperoxy-FAD intermediate that is crucial for substrate oxygenatio n (Alfieri et al, 2008;Orru et al, 2010), S195L mutation may destabilise the NADP + binding and accelerate the decay of the intermediate, which is in good agreement with the dramatically reduced kcat for TMA ( Table 2).…”

Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

“…Thus, the analog does not bind in a conformation optimal for the stabilization of the oxygenated flavin intermediates. Similar effects were observed by mutating residues that interact with the amide group of the nicotinamide ring in FMO (35). Because NADP(H) is expected to play a similar role in SidA as reported for FMO, it is reasonable to assume that a similar effect occurs in SidA.…”

“…In addition, we also showed that reduction of the flavin results in interaction of the carbonyl oxygen of the amide group of NADP ϩ with N5 of the flavin. This interaction is believed to be essential in preventing the release of hydrogen peroxide, as demonstrated in other flavin-dependent reactions (14,29,35,36).…”