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W2146703417 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 hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications. 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 hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications. IntroductionFlavin-containing monooxygenases (FMOs) 3The abbreviations used are: FMOflavin-containing monooxygenaseFMO3isozyme 3 of human FMOAPADP3-acetylpyridine adenine dinucleotidemFMOFMO from Methylophaga sp. strain SK1TMAUtrimethylaminuria. form a large family of enzymes present almost ubiquitously among living organisms (1Cashman J.R. Zhang J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 65-100Crossref PubMed Scopus (249) Google Scholar, 2Krueger S.K. Williams D.E. Pharmacol. Ther. 2005; 106: 3573-3587Crossref Scopus (423) Google Scholar, 3Phillips I.R. Shephard E.A. Trends Pharmacol. Sci. 2008; 29: 294-301Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). They are involved in diverse biological processes such as the biosynthesis of various natural products (4Schlaich N.L. Trends Plant Sci. 2007; 12: 412-448Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) 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 (5Al-Waiz M. Ayesh R. Mitchell S.C. Idle J.R. Smith R.L. Lancet. 1987; 1: 634-635Abstract PubMed Scopus (39) Google Scholar, 6Dolphin C.T. Janmohamed A. Smith R.L. Shephard E.A. Phillips I.R. Nat. Genet. 1997; 17: 491-494Crossref PubMed Scopus (218) Google Scholar).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 (7Ziegler D.M. Drug Metab. Rev. 2002; 34: 503-511Crossref PubMed Scopus (163) Google Scholar) (Fig. 1A). At the heart of this very complex reaction is the stabilization of the flavin-hydroperoxide adduct resulting from the reaction of dioxygen with the two-electron reduced FAD (Fig. 1A). Equally essential is the role of NADP(H). This cofactor not only acts as the electron donor that reduces the flavin but it directly takes part also in formation/stabilization of the flavin-hydroperoxide. This notion is best illustrated by the fact that using alternative electron donors (e.g. dithionite) in place of NADPH does not support catalysis because the reaction of dioxygen with the artificially reduced flavin generates hydrogen peroxide rather than the stable flavin-hydroperoxide needed for monooxygenation (8Beaty N.B. Ballou D.P. J. Biol. Chem. 1980; 255: 3817-3819Abstract Full Text PDF PubMed Google Scholar, 9Beaty N.B. Ballou D.P. J. Biol. Chem. 1981; 256: 4611-4618Abstract Full Text PDF PubMed Google Scholar, 10Beaty N.B. Ballou D.P. J. Biol. Chem. 1981; 256: 4619-4625Abstract Full Text PDF PubMed Google Scholar). In the cellular milieu, FMOs are thought to be mainly in the flavin-hydroperoxide state, ready to attack a suitable substrate (the so-called “cocked gun”) (11Poulsen L.L. Ziegler D.M. J. Biol. Chem. 1979; 254: 6449-6455Abstract Full Text PDF PubMed Google Scholar).The peculiar catalytic role of NADP(H) in oxygenation makes FMOs a very attractive system to study the more general problem of oxygen reactivity in flavoenzymes. Because it has been established that the nicotinamide cofactor remains bound to enzyme during oxygenation, NADP(H) can be exploited as a probe to gain insight into the reaction of dioxygen with the reduced flavin and to interrogate the elements that promote the stabilization of the crucial flavin-hydroperoxide. This concept represents the starting point of our study that builds on the previous analysis of the crystal structure and basic biochemical properties of the FMO from Methylophaga sp. strain SK1 (mFMO, Fig. 1B) (12Choi H.S. Kim J.K. Cho E.H. Kim Y.C. Kim J.I. Kim S.W. Biochem. Biophys. Res. Commun. 2003; 306: 930-936Crossref PubMed Scopus (84) Google Scholar). This bacterial enzyme shares many features with the human FMO3 including a considerable degree of sequence identity (33%), a similar substrate specificity, and the ability to form a stable flavin-hydroperoxide intermediate (13Alfieri A. Malito E. Orru R. Fraaije M.W. Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 2008; 195: 6572-6577Crossref Scopus (113) Google Scholar). At the same time, mFMO is better suited for detailed structural and enzymological investigations because it is a soluble protein (i.e. not membrane-associated). The aim of our study is 2-fold: (i) to gain insight into the astonishing properties of FMOs that combine in a single active center two cofactors and a complex sequence of catalytic steps that lead to the formation of an “activated-form” of dioxygen (flavin-hydroperoxide, Fig. 1A), and (ii) to translate this knowledge into a molecular rational for the effects of mutations found in patients affected by TMAU. For these purposes, we have probed the mFMO catalytic properties by altering both the active site amino acids by site directed mutagenesis and the NADP(H) substrate by using analogues of this cofactor. Such a “double-engineering” strategy has revealed that very fine details in the stereochemical and hydrogen-bonding environment around the flavin determine the efficiency of the reaction with dioxygen and the nature of the resulting products.DISCUSSIONThe specific objective of our study was to investigate the reaction of FMO with dioxygen using site-directed mutagenesis and NADP(H) analogues as dissecting tools. The general aim was to improve our knowledge of the molecular basis of TMAU and to advance our current understanding of the oxygen reactivity in flavoenzymes. Along these lines, we can draw several conclusions.The analysis of the Asn-78 mutants indicates that at least some of the TMAU-causing mutations target the Achilles heel of the enzyme: the intrinsic “fragility” of the crucial flavin-hydroperoxide intermediate, which, if not appropriately stabilized, causes the conversion of the enzyme into an oxidase.In this scenario, the fact that the devil is in the detail is best illustrated by the N78S variant (Figs. 2A and 3A). This mutation does not affect the steric hindrance and hydrophilic nature of the side chain and does not cause any detectable change in the active site conformation. Nevertheless, it fully inactivates the enzyme. Evidently, the fine details in the environment of the C4a atom (Fig. 1A) can make a tremendous difference in the flavin reactivity. In this case, it appears that a Ser side chain in position 78 is unable to provide the precisely positioned H-bond partner required to stabilize the flavin-hydroperoxide, whose terminal oxygen can accept and donate H-bonds (Figs. 1A and 3A). Therefore, the intermediate does not form at all or, more likely, it decays too rapidly to sustain monooxygenation (Fig. 2B). As a result, the mutant enzyme retains (albeit less efficient) reactivity with oxygen but only to function as oxidase and not as monooxygenase (Table 1).The N78D mutant illustrates the role of electrostatics. This isosteric mutation positions a negative charge close to the N5 with to a 30-fold reduction in the kox (Table 1). As observed for other flavoenzymes, it is likely that this mutation disfavors the first step of the oxygen reaction; the one-electron transfer leading to the formation of flavin semiquinone-superoxide caged radical pair that subsequently generates the flavin-hydroperoxide and/or hydrogen peroxide (24Mattevi A. Trends Biochem. Sci. 2006; 31: 276-283Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 25Massey V. J. Biol. Chem. 1994; 269: 22459-22462Abstract Full Text PDF PubMed Google Scholar).Monooxygenation requires that NADP+ binds in the proper conformation as outlined by the study of the N78D mutant and the experiments with the APADP+ analog (Figs. 3B and 4B). The role of NADP+ is manifold: (i) together with Asn-78, it creates the niche for oxygen binding in front of the flavin C4a atom, (ii) its ribose 2′-hydroxyl group provides a potential H-bonding partner for the flavin-hydroperoxide atoms (13Alfieri A. Malito E. Orru R. Fraaije M.W. Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 2008; 195: 6572-6577Crossref Scopus (113) Google Scholar), and (iii) it forms key H-bond interactions with the flavin N5 and O4 atoms (Fig. 1B). This latter feature should be emphasized because it can have a crucial role. It is known that protection of the flavin N5 atom can enormously enhance the stability of the flavin-hydroperoxide to the point that the reaction of oxygen with protein-free reduced N5-alkylriboflavin forms a very stable and biocatalytically useful hydroperoxide derivative (26Kemal C. Chan T.W. Bruice R.C. J. Am. Chem. Soc. 1977; 99: 7272-7286Crossref PubMed Scopus (144) Google Scholar, 27Imada Y. Iida H. Murahashi S. Naota T. Angew. Chem. Int. Ed. Engl. 2005; 44: 1704-1706Crossref PubMed Scopus (141) Google Scholar). Although the N5 atom of the two-electron reduced flavin has a very high pKa value (around 20) (28Macheroux P. Ghisla S. Sanner C. Rüterjans H. Müller F. BMC Biochem. 2005; 6: 26Crossref PubMed Scopus (42) Google Scholar), adequate protection of this atom is probably needed to inhibit rapid proton exchange with the solvent which may concomitantly trigger decay of the flavin-hydroperoxide. Likewise, protection of the N5 may also prevent that this atom directly takes part in the oxygen reaction for example by acting as hydrogen or proton donor.The comparison of the NADP+ and APADP+ complexes (Fig. 4C) provides a glimpse of the likely mode of binding of the organic substrate. Indeed, as visualized by Fig. 4C, the “out” position of the APADP+ pyridine moiety corresponds to the location expected for an organic substrate (for example indole or nicotine) to be monooxygenated by the enzyme. The substrate promiscuity of FMOs, which act on a variety of diverse molecules, seems to inevitably expose these enzymes to the risk of binding the pyridine ring of the NADP(H) ligand in the catalytically incorrect positions as indicated by the mutagenesis and NADP-analog studies. In this context, it is remarkable that the mFMO appears to use a Tyr residue as a backdoor that sandwiches the nicotinamide in the H-bonding position with N5 (Fig. 3C).The more general conclusion from this analysis is that the two alternative pathways (oxidation versus monooxygenation) along the oxygen reaction can be predicted to have very similar energy barriers so that subtle alterations in the balance of interactions around the C4a-N5 atoms of the flavin can drastically alter the outcome of the reaction of the reduced enzyme with dioxygen. This will be a crucial aspect to be considered in the design and development of FMOs and related monooxygenases for biocatalytic application. IntroductionFlavin-containing monooxygenases (FMOs) 3The abbreviations used are: FMOflavin-containing monooxygenaseFMO3isozyme 3 of human FMOAPADP3-acetylpyridine adenine dinucleotidemFMOFMO from Methylophaga sp. strain SK1TMAUtrimethylaminuria. form a large family of enzymes present almost ubiquitously among living organisms (1Cashman J.R. Zhang J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 65-100Crossref PubMed Scopus (249) Google Scholar, 2Krueger S.K. Williams D.E. Pharmacol. Ther. 2005; 106: 3573-3587Crossref Scopus (423) Google Scholar, 3Phillips I.R. Shephard E.A. Trends Pharmacol. Sci. 2008; 29: 294-301Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). They are involved in diverse biological processes such as the biosynthesis of various natural products (4Schlaich N.L. Trends Plant Sci. 2007; 12: 412-448Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) 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 (5Al-Waiz M. Ayesh R. Mitchell S.C. Idle J.R. Smith R.L. Lancet. 1987; 1: 634-635Abstract PubMed Scopus (39) Google Scholar, 6Dolphin C.T. Janmohamed A. Smith R.L. Shephard E.A. Phillips I.R. Nat. Genet. 1997; 17: 491-494Crossref PubMed Scopus (218) Google Scholar).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 (7Ziegler D.M. Drug Metab. Rev. 2002; 34: 503-511Crossref PubMed Scopus (163) Google Scholar) (Fig. 1A). At the heart of this very complex reaction is the stabilization of the flavin-hydroperoxide adduct resulting from the reaction of dioxygen with the two-electron reduced FAD (Fig. 1A). Equally essential is the role of NADP(H). This cofactor not only acts as the electron donor that reduces the flavin but it directly takes part also in formation/stabilization of the flavin-hydroperoxide. This notion is best illustrated by the fact that using alternative electron donors (e.g. dithionite) in place of NADPH does not support catalysis because the reaction of dioxygen with the artificially reduced flavin generates hydrogen peroxide rather than the stable flavin-hydroperoxide needed for monooxygenation (8Beaty N.B. Ballou D.P. J. Biol. Chem. 1980; 255: 3817-3819Abstract Full Text PDF PubMed Google Scholar, 9Beaty N.B. Ballou D.P. J. Biol. Chem. 1981; 256: 4611-4618Abstract Full Text PDF PubMed Google Scholar, 10Beaty N.B. Ballou D.P. J. Biol. Chem. 1981; 256: 4619-4625Abstract Full Text PDF PubMed Google Scholar). In the cellular milieu, FMOs are thought to be mainly in the flavin-hydroperoxide state, ready to attack a suitable substrate (the so-called “cocked gun”) (11Poulsen L.L. Ziegler D.M. J. Biol. Chem. 1979; 254: 6449-6455Abstract Full Text PDF PubMed Google Scholar).The peculiar catalytic role of NADP(H) in oxygenation makes FMOs a very attractive system to study the more general problem of oxygen reactivity in flavoenzymes. Because it has been established that the nicotinamide cofactor remains bound to enzyme during oxygenation, NADP(H) can be exploited as a probe to gain insight into the reaction of dioxygen with the reduced flavin and to interrogate the elements that promote the stabilization of the crucial flavin-hydroperoxide. This concept represents the starting point of our study that builds on the previous analysis of the crystal structure and basic biochemical properties of the FMO from Methylophaga sp. strain SK1 (mFMO, Fig. 1B) (12Choi H.S. Kim J.K. Cho E.H. Kim Y.C. Kim J.I. Kim S.W. Biochem. Biophys. Res. Commun. 2003; 306: 930-936Crossref PubMed Scopus (84) Google Scholar). This bacterial enzyme shares many features with the human FMO3 including a considerable degree of sequence identity (33%), a similar substrate specificity, and the ability to form a stable flavin-hydroperoxide intermediate (13Alfieri A. Malito E. Orru R. Fraaije M.W. Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 2008; 195: 6572-6577Crossref Scopus (113) Google Scholar). At the same time, mFMO is better suited for detailed structural and enzymological investigations because it is a soluble protein (i.e. not membrane-associated). The aim of our study is 2-fold: (i) to gain insight into the astonishing properties of FMOs that combine in a single active center two cofactors and a complex sequence of catalytic steps that lead to the formation of an “activated-form” of dioxygen (flavin-hydroperoxide, Fig. 1A), and (ii) to translate this knowledge into a molecular rational for the effects of mutations found in patients affected by TMAU. For these purposes, we have probed the mFMO catalytic properties by altering both the active site amino acids by site directed mutagenesis and the NADP(H) substrate by using analogues of this cofactor. Such a “double-engineering” strategy has revealed that very fine details in the stereochemical and hydrogen-bonding environment around the flavin determine the efficiency of the reaction with dioxygen and the nature of the resulting products. Flavin-containing monooxygenases (FMOs) 3The abbreviations used are: FMOflavin-containing monooxygenaseFMO3isozyme 3 of human FMOAPADP3-acetylpyridine adenine dinucleotidemFMOFMO from Methylophaga sp. strain SK1TMAUtrimethylaminuria. form a large family of enzymes present almost ubiquitously among living organisms (1Cashman J.R. Zhang J. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 65-100Crossref PubMed Scopus (249) Google Scholar, 2Krueger S.K. Williams D.E. Pharmacol. Ther. 2005; 106: 3573-3587Crossref Scopus (423) Google Scholar, 3Phillips I.R. Shephard E.A. Trends Pharmacol. Sci. 2008; 29: 294-301Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). They are involved in diverse biological processes such as the biosynthesis of various natural products (4Schlaich N.L. Trends Plant Sci. 2007; 12: 412-448Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) 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 (5Al-Waiz M. Ayesh R. Mitchell S.C. Idle J.R. Smith R.L. Lancet. 1987; 1: 634-635Abstract PubMed Scopus (39) Google Scholar, 6Dolphin C.T. Janmohamed A. Smith R.L. Shephard E.A. Phillips I.R. Nat. Genet. 1997; 17: 491-494Crossref PubMed Scopus (218) Google Scholar). flavin-containing monooxygenase isozyme 3 of human FMO 3-acetylpyridine adenine dinucleotide FMO from Methylophaga sp. strain SK1 trimethylaminuria. 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 (7Ziegler D.M. Drug Metab. Rev. 2002; 34: 503-511Crossref PubMed Scopus (163) Google Scholar) (Fig. 1A). At the heart of this very complex reaction is the stabilization of the flavin-hydroperoxide adduct resulting from the reaction of dioxygen with the two-electron reduced FAD (Fig. 1A). Equally essential is the role of NADP(H). This cofactor not only acts as the electron donor that reduces the flavin but it directly takes part also in formation/stabilization of the flavin-hydroperoxide. This notion is best illustrated by the fact that using alternative electron donors (e.g. dithionite) in place of NADPH does not support catalysis because the reaction of dioxygen with the artificially reduced flavin generates hydrogen peroxide rather than the stable flavin-hydroperoxide needed for monooxygenation (8Beaty N.B. Ballou D.P. J. Biol. Chem. 1980; 255: 3817-3819Abstract Full Text PDF PubMed Google Scholar, 9Beaty N.B. Ballou D.P. J. Biol. Chem. 1981; 256: 4611-4618Abstract Full Text PDF PubMed Google Scholar, 10Beaty N.B. Ballou D.P. J. Biol. Chem. 1981; 256: 4619-4625Abstract Full Text PDF PubMed Google Scholar). In the cellular milieu, FMOs are thought to be mainly in the flavin-hydroperoxide state, ready to attack a suitable substrate (the so-called “cocked gun”) (11Poulsen L.L. Ziegler D.M. J. Biol. Chem. 1979; 254: 6449-6455Abstract Full Text PDF PubMed Google Scholar). The peculiar catalytic role of NADP(H) in oxygenation makes FMOs a very attractive system to study the more general problem of oxygen reactivity in flavoenzymes. Because it has been established that the nicotinamide cofactor remains bound to enzyme during oxygenation, NADP(H) can be exploited as a probe to gain insight into the reaction of dioxygen with the reduced flavin and to interrogate the elements that promote the stabilization of the crucial flavin-hydroperoxide. This concept represents the starting point of our study that builds on the previous analysis of the crystal structure and basic biochemical properties of the FMO from Methylophaga sp. strain SK1 (mFMO, Fig. 1B) (12Choi H.S. Kim J.K. Cho E.H. Kim Y.C. Kim J.I. Kim S.W. Biochem. Biophys. Res. Commun. 2003; 306: 930-936Crossref PubMed Scopus (84) Google Scholar). This bacterial enzyme shares many features with the human FMO3 including a considerable degree of sequence identity (33%), a similar substrate specificity, and the ability to form a stable flavin-hydroperoxide intermediate (13Alfieri A. Malito E. Orru R. Fraaije M.W. Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 2008; 195: 6572-6577Crossref Scopus (113) Google Scholar). At the same time, mFMO is better suited for detailed structural and enzymological investigations because it is a soluble protein (i.e. not membrane-associated). The aim of our study is 2-fold: (i) to gain insight into the astonishing properties of FMOs that combine in a single active center two cofactors and a complex sequence of catalytic steps that lead to the formation of an “activated-form” of dioxygen (flavin-hydroperoxide, Fig. 1A), and (ii) to translate this knowledge into a molecular rational for the effects of mutations found in patients affected by TMAU. For these purposes, we have probed the mFMO catalytic properties by altering both the active site amino acids by site directed mutagenesis and the NADP(H) substrate by using analogues of this cofactor. Such a “double-engineering” strategy has revealed that very fine details in the stereochemical and hydrogen-bonding environment around the flavin determine the efficiency of the reaction with dioxygen and the nature of the resulting products. DISCUSSIONThe specific objective of our study was to investigate the reaction of FMO with dioxygen using site-directed mutagenesis and NADP(H) analogues as dissecting tools. The general aim was to improve our knowledge of the molecular basis of TMAU and to advance our current understanding of the oxygen reactivity in flavoenzymes. Along these lines, we can draw several conclusions.The analysis of the Asn-78 mutants indicates that at least some of the TMAU-causing mutations target the Achilles heel of the enzyme: the intrinsic “fragility” of the crucial flavin-hydroperoxide intermediate, which, if not appropriately stabilized, causes the conversion of the enzyme into an oxidase.In this scenario, the fact that the devil is in the detail is best illustrated by the N78S variant (Figs. 2A and 3A). This mutation does not affect the steric hindrance and hydrophilic nature of the side chain and does not cause any detectable change in the active site conformation. Nevertheless, it fully inactivates the enzyme. Evidently, the fine details in the environment of the C4a atom (Fig. 1A) can make a tremendous difference in the flavin reactivity. In this case, it appears that a Ser side chain in position 78 is unable to provide the precisely positioned H-bond partner required to stabilize the flavin-hydroperoxide, whose terminal oxygen can accept and donate H-bonds (Figs. 1A and 3A). Therefore, the intermediate does not form at all or, more likely, it decays too rapidly to sustain monooxygenation (Fig. 2B). As a result, the mutant enzyme retains (albeit less efficient) reactivity with oxygen but only to function as oxidase and not as monooxygenase (Table 1).The N78D mutant illustrates the role of electrostatics. This isosteric mutation positions a negative charge close to the N5 with to a 30-fold reduction in the kox (Table 1). As observed for other flavoenzymes, it is likely that this mutation disfavors the first step of the oxygen reaction; the one-electron transfer leading to the formation of flavin semiquinone-superoxide caged radical pair that subsequently generates the flavin-hydroperoxide and/or hydrogen peroxide (24Mattevi A. Trends Biochem. Sci. 2006; 31: 276-283Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 25Massey V. J. Biol. Chem. 1994; 269: 22459-22462Abstract Full Text PDF PubMed Google Scholar).Monooxygenation requires that NADP+ binds in the proper conformation as outlined by the study of the N78D mutant and the experiments with the APADP+ analog (Figs. 3B and 4B). The role of NADP+ is manifold: (i) together with Asn-78, it creates the niche for oxygen binding in front of the flavin C4a atom, (ii) its ribose 2′-hydroxyl group provides a potential H-bonding partner for the flavin-hydroperoxide atoms (13Alfieri A. Malito E. Orru R. Fraaije M.W. Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 2008; 195: 6572-6577Crossref Scopus (113) Google Scholar), and (iii) it forms key H-bond interactions with the flavin N5 and O4 atoms (Fig. 1B). This latter feature should be emphasized because it can have a crucial role. It is known that protection of the flavin N5 atom can enormously enhance the stability of the flavin-hydroperoxide to the point that the reaction of oxygen with protein-free reduced N5-alkylriboflavin forms a very stable and biocatalytically useful hydroperoxide derivative (26Kemal C. Chan T.W. Bruice R.C. J. Am. Chem. Soc. 1977; 99: 7272-7286Crossref PubMed Scopus (144) Google Scholar, 27Imada Y. Iida H. Murahashi S. Naota T. Angew. Chem. Int. Ed. Engl. 2005; 44: 1704-1706Crossref PubMed Scopus (141) Google Scholar). Although the N5 atom of the two-electron reduced flavin has a very high pKa value (around 20) (28Macheroux P. Ghisla S. Sanner C. Rüterjans H. Müller F. BMC Biochem. 2005; 6: 26Crossref PubMed Scopus (42) Google Scholar), adequate protection of this atom is probably needed to inhibit rapid proton exchange with the solvent which may concomitantly trigger decay of the flavin-hydroperoxide. Likewise, protection of the N5 may also prevent that this atom directly takes part in the oxygen reaction for example by acting as hydrogen or proton donor.The comparison of the NADP+ and APADP+ complexes (Fig. 4C) provides a glimpse of the likely mode of binding of the organic substrate. Indeed, as visualized by Fig. 4C, the “out” position of the APADP+ pyridine moiety corresponds to the location expected for an organic substrate (for example indole or nicotine) to be monooxygenated by the enzyme. The substrate promiscuity of FMOs, which act on a variety of diverse molecules, seems to inevitably expose these enzymes to the risk of binding the pyridine ring of the NADP(H) ligand in the catalytically incorrect positions as indicated by the mutagenesis and NADP-analog studies. In this context, it is remarkable that the mFMO appears to use a Tyr residue as a backdoor that sandwiches the nicotinamide in the H-bonding position with N5 (Fig. 3C).The more general conclusion from this analysis is that the two alternative pathways (oxidation versus monooxygenation) along the oxygen reaction can be predicted to have very similar energy barriers so that subtle alterations in the balance of interactions around the C4a-N5 atoms of the flavin can drastically alter the outcome of the reaction of the reduced enzyme with dioxygen. This will be a crucial aspect to be considered in the design and development of FMOs and related monooxygenases for biocatalytic application. The specific objective of our study was to investigate the reaction of FMO with dioxygen using site-directed mutagenesis and NADP(H) analogues as dissecting tools. The general aim was to improve our knowledge of the molecular basis of TMAU and to advance our current understanding of the oxygen reactivity in flavoenzymes. Along these lines, we can draw several conclusions. The analysis of the Asn-78 mutants indicates that at least some of the TMAU-causing mutations target the Achilles heel of the enzyme: the intrinsic “fragility” of the crucial flavin-hydroperoxide intermediate, which, if not appropriately stabilized, causes the conversion of the enzyme into an oxidase. In this scenario, the fact that the devil is in the detail is best illustrated by the N78S variant (Figs. 2A and 3A). This mutation does not affect the steric hindrance and hydrophilic nature of the side chain and does not cause any detectable change in the active site conformation. Nevertheless, it fully inactivates the enzyme. Evidently, the fine details in the environment of the C4a atom (Fig. 1A) can make a tremendous difference in the flavin reactivity. In this case, it appears that a Ser side chain in position 78 is unable to provide the precisely positioned H-bond partner required to stabilize the flavin-hydroperoxide, whose terminal oxygen can accept and donate H-bonds (Figs. 1A and 3A). Therefore, the intermediate does not form at all or, more likely, it decays too rapidly to sustain monooxygenation (Fig. 2B). As a result, the mutant enzyme retains (albeit less efficient) reactivity with oxygen but only to function as oxidase and not as monooxygenase (Table 1). The N78D mutant illustrates the role of electrostatics. This isosteric mutation positions a negative charge close to the N5 with to a 30-fold reduction in the kox (Table 1). As observed for other flavoenzymes, it is likely that this mutation disfavors the first step of the oxygen reaction; the one-electron transfer leading to the formation of flavin semiquinone-superoxide caged radical pair that subsequently generates the flavin-hydroperoxide and/or hydrogen peroxide (24Mattevi A. Trends Biochem. Sci. 2006; 31: 276-283Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 25Massey V. J. Biol. Chem. 1994; 269: 22459-22462Abstract Full Text PDF PubMed Google Scholar). Monooxygenation requires that NADP+ binds in the proper conformation as outlined by the study of the N78D mutant and the experiments with the APADP+ analog (Figs. 3B and 4B). The role of NADP+ is manifold: (i) together with Asn-78, it creates the niche for oxygen binding in front of the flavin C4a atom, (ii) its ribose 2′-hydroxyl group provides a potential H-bonding partner for the flavin-hydroperoxide atoms (13Alfieri A. Malito E. Orru R. Fraaije M.W. Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 2008; 195: 6572-6577Crossref Scopus (113) Google Scholar), and (iii) it forms key H-bond interactions with the flavin N5 and O4 atoms (Fig. 1B). This latter feature should be emphasized because it can have a crucial role. It is known that protection of the flavin N5 atom can enormously enhance the stability of the flavin-hydroperoxide to the point that the reaction of oxygen with protein-free reduced N5-alkylriboflavin forms a very stable and biocatalytically useful hydroperoxide derivative (26Kemal C. Chan T.W. Bruice R.C. J. Am. Chem. Soc. 1977; 99: 7272-7286Crossref PubMed Scopus (144) Google Scholar, 27Imada Y. Iida H. Murahashi S. Naota T. Angew. Chem. Int. Ed. Engl. 2005; 44: 1704-1706Crossref PubMed Scopus (141) Google Scholar). Although the N5 atom of the two-electron reduced flavin has a very high pKa value (around 20) (28Macheroux P. Ghisla S. Sanner C. Rüterjans H. Müller F. BMC Biochem. 2005; 6: 26Crossref PubMed Scopus (42) Google Scholar), adequate protection of this atom is probably needed to inhibit rapid proton exchange with the solvent which may concomitantly trigger decay of the flavin-hydroperoxide. Likewise, protection of the N5 may also prevent that this atom directly takes part in the oxygen reaction for example by acting as hydrogen or proton donor. The comparison of the NADP+ and APADP+ complexes (Fig. 4C) provides a glimpse of the likely mode of binding of the organic substrate. Indeed, as visualized by Fig. 4C, the “out” position of the APADP+ pyridine moiety corresponds to the location expected for an organic substrate (for example indole or nicotine) to be monooxygenated by the enzyme. The substrate promiscuity of FMOs, which act on a variety of diverse molecules, seems to inevitably expose these enzymes to the risk of binding the pyridine ring of the NADP(H) ligand in the catalytically incorrect positions as indicated by the mutagenesis and NADP-analog studies. In this context, it is remarkable that the mFMO appears to use a Tyr residue as a backdoor that sandwiches the nicotinamide in the H-bonding position with N5 (Fig. 3C). The more general conclusion from this analysis is that the two alternative pathways (oxidation versus monooxygenation) along the oxygen reaction can be predicted to have very similar energy barriers so that subtle alterations in the balance of interactions around the C4a-N5 atoms of the flavin can drastically alter the outcome of the reaction of the reduced enzyme with dioxygen. This will be a crucial aspect to be considered in the design and development of FMOs and related monooxygenases for biocatalytic application. We thank Dr. Andrea Alfieri for contributions in the earlier stages of the project." @default.
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W2146703417 title "Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase" @default.
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