Unifying and versatile features of flavin-dependent monooxygenases: Diverse catalysis by a common C4a-(hydro)peroxyflavin

Flavin-dependent monooxygenases (FDMOs) are known for their remarkable versatility and for their crucial roles in various biological processes and applications. Extensive research has been conducted to explore the structural and functional relationships of FDMOs. The majority of reported FDMOs utilize C4a-(hydro)peroxyflavin as a reactive intermediate to incorporate an oxygen atom into a wide range of compounds. This review discusses and analyzes recent advancements in our understanding of the structural and mechanistic features governing the enzyme functions. State-of-the-art discoveries related to common and distinct structural properties governing the catalytic versatility of the C4a-(hydro)peroxyflavin intermediate in selected FDMOs are discussed. Specifically, mechanisms of hydroxylation, dehalogenation, halogenation, and light-emitting reactions by FDMOs are highlighted. We also provide new analysis based on the structural and mechanistic features of these enzymes to gain insights into how the same intermediate can be harnessed to perform a wide variety of reactions. Challenging questions to obtain further breakthroughs in the understanding of FDMOs are also proposed. Flavin-dependent monooxygenases (FDMOs) are known for their remarkable versatility and for their crucial roles in various biological processes and applications. Extensive research has been conducted to explore the structural and functional relationships of FDMOs. The majority of reported FDMOs utilize C4a-(hydro)peroxyflavin as a reactive intermediate to incorporate an oxygen atom into a wide range of compounds. This review discusses and analyzes recent advancements in our understanding of the structural and mechanistic features governing the enzyme functions. State-of-the-art discoveries related to common and distinct structural properties governing the catalytic versatility of the C4a-(hydro)peroxyflavin intermediate in selected FDMOs are discussed. Specifically, mechanisms of hydroxylation, dehalogenation, halogenation, and light-emitting reactions by FDMOs are highlighted. We also provide new analysis based on the structural and mechanistic features of these enzymes to gain insights into how the same intermediate can be harnessed to perform a wide variety of reactions. Challenging questions to obtain further breakthroughs in the understanding of FDMOs are also proposed. Flavin-dependent enzymes utilize flavin derivatives—mostly FMN or flavin adenine dinucleotide (FAD)—which are derived from vitamin B2, as cofactors or substrates. These flavin cofactors exhibit remarkable versatility, as they can undergo one- or two-electron transfers, allowing the enzymes to adopt multiple redox states (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 2Toplak M. Teufel R. Three rings to rule them all: how versatile flavoenzymes orchestrate the structural diversification of natural products.Biochemistry. 2022; 61: 47-56Google Scholar, 3Pimviriyakul P. Chaiyen P. Chapter one - overview of flavin-dependent enzymes.in: Chaiyen P. Tamanoi F. The Enzymes. Academic Press, Cambridge, MA2020: 1-36Google Scholar). The ability of flavins to exist in a transient radical state (i.e. flavin semiquinone) either through a natural redox cycle (reduced by a substrate) or light activation enables a wide range of challenging reactivities such as oxygen activation, C-C-bond formation, C-C-bond cleavage, and C-N bond formation (4Sandoval B.A. Meichan A.J. Hyster T.K. Enantioselective hydrogen atom transfer: discovery of catalytic promiscuity in flavin-dependent 'ene'-reductases.J. Am. Chem. Soc. 2017; 139: 11313-11316Google Scholar, 5Foja R. Walter A. Jandl C. Thyrhaug E. Hauer J. Storch G. Reduced molecular flavins as single-electron reductants after photoexcitation.J. Am. Chem. Soc. 2022; 144: 4721-4726Google Scholar, 6Black M.J. Biegasiewicz K.F. Meichan A.J. Oblinsky D.G. Kudisch B. Scholes G.D. et al.Asymmetric redox-neutral radical cyclization catalysed by flavin-dependent 'ene'-reductases.Nat. Chem. 2020; 12: 71-75Google Scholar, 7Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37: 373-380Google Scholar, 8Zhang Z. Feng J. Yang C. Cui H. Harrison W. Zhong D. et al.Photoenzymatic enantioselective intermolecular radical hydroamination.Nat. Catalysis. 2023; 6: 687-694Google Scholar). Flavin-dependent enzymes play pivotal roles in diverse catalytic reactions and biological processes involved in mostly redox metabolisms such as xenobiotic detoxification, biosynthetic pathways in plants and biosynthesis of microbial secondary metabolites, neural development in humans, and light-emitting reactions in bacteria. Non-redox reactions such as galactofuranose synthesis in bacteria and fungi can also be catalyzed by flavin-dependent enzymes (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 2Toplak M. Teufel R. Three rings to rule them all: how versatile flavoenzymes orchestrate the structural diversification of natural products.Biochemistry. 2022; 61: 47-56Google Scholar, 9Joosten V. van Berkel W.J.H. Flavoenzymes.Curr. Opin. Chem. Biol. 2007; 11: 195-202Google Scholar, 10van Berkel W.J.H. Flavoenzymes, Chemistry of, Wiley Encyclopedia of Chemical Biology. Wiley, Hoboken, NJ2008Google Scholar, 11Palfey B.A. McDonald C.A. Control of catalysis in flavin-dependent monooxygenases.Arch. Biochem. Biophys. 2010; 493: 26-36Google Scholar, 12Tanner J.J. Boechi L. Andrew McCammon J. Sobrado P. Structure, mechanism, and dynamics of UDP-galactopyranose mutase.Arch. Biochem. Biophys. 2014; 544: 128-141Google Scholar). Flavin-dependent enzymes, especially the flavin-dependent monooxygenases (FDMOs), have drawn significant attention in the fields of biochemistry and biotechnology due to their involvement in a broad spectrum of catalytic reactions. FDMOs function by incorporating a single atom of molecular oxygen into their substrates. These enzymes have great potential for use in various applications including the biosynthesis of valuable chemicals, as well as in drug metabolisms, biodetoxification, bioremediation, and biosensors (3Pimviriyakul P. Chaiyen P. Chapter one - overview of flavin-dependent enzymes.in: Chaiyen P. Tamanoi F. The Enzymes. Academic Press, Cambridge, MA2020: 1-36Google Scholar, 13Ceccoli R. Bianchi D. Rial D. Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications.Front. Microbiol. 2014; 5: 25Google Scholar, 14Reis R.A.G. Li H. Johnson M. Sobrado P. New frontiers in flavin-dependent monooxygenases.Arch. Biochem. Biophys. 2021; 699108765Google Scholar). FDMOs comprise a diverse group of enzymes that exhibit a wide variety of structures and functions. Based on the protein components, these enzymes can be divided into two main types: single-component and two-component monooxygenases. Single-component monooxygenases bind flavin constitutively; flavin reduction by NAD or NAD(P)H and substrate monooxygenation occur at the same active site. In contrast, two-component monooxygenases require a flavin reductase to generate a reduced flavin (15Chenprakhon P. Wongnate T. Chaiyen P. Monooxygenation of aromatic compounds by flavin-dependent monooxygenases.Protein Sci. 2019; 28: 8-29Google Scholar, 16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar). The reduced flavin is then transferred (can occur via diffusion) from a flavin reductase to the monooxygenase for subsequent oxygen activation and substrate monooxygenation (17Sucharitakul J. Tinikul R. Chaiyen P. Mechanisms of reduced flavin transfer in the two-component flavin-dependent monooxygenases.Arch. Biochem. Biophys. 2014; 555-556: 33-46Google Scholar, 18Sucharitakul J. Phongsak T. Entsch B. Svasti J. Chaiyen P. Ballou D.P. Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase.Biochemistry. 2007; 46: 8611-8623Google Scholar). FDMOs can be categorized into eight groups (groups A-H) based on their structural characteristics and functional properties (16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar, 19Mascotti M.L. Juri Ayub M. Furnham N. Thornton J.M. Laskowski R.A. Chopping and changing: the evolution of the flavin-dependent monooxygenases.J. Mol. Biol. 2016; 428: 3131-3146Google Scholar). They activate oxygen and generate reactive intermediates through flavin oxygenation reactions, forming either C4a-(hydro)peroxyflavin or flavin N5-peroxide as oxygenating reagents (Fig. 1) (7Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37: 373-380Google Scholar, 20Toplak M. Matthews A. Teufel R. The devil is in the details: the chemical basis and mechanistic versatility of flavoprotein monooxygenases.Arch. Biochem. Biophys. 2021; 698108732Google Scholar, 21Teufel R. Stull F.W. Meehan M.J. Michaudel Q. Dorrestein P.C. Palfey B.A. et al.Biochemical establishment and characterization of EncM's flavin-N5-oxide cofactor.J. Am. Chem. Soc. 2015; 137 25: 8078-8085Google Scholar). Among the known FDMOs, most of the enzymes form C4a-(hydro)peroxyflavin, which plays a critical role in FDMOs as an oxygen donator via nucleophilic or electrophilic reactions. Herein, we focus on the reactions of FDMOs that generate C4a-(hydro)peroxyflavin as an intermediate. This intermediate allows FDMOs to catalyze a wide range of reactions including hydroxylation, Baeyer–Villiger oxidation, sulfoxidation, epoxidation, denitration, dehalogenation, halogenation reactions, and light emission (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar, 22van Berkel W.J.H. Kamerbeek N.M. Fraaije M.W. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts.J. Biotechnol. 2006; 124: 670-689Google Scholar). Understanding how different FDMOs control the remarkable diversity of C4a-(hydro)peroxyflavin would allow these enzymes to be engineered for a variety of impactful applications. This review thus highlights the current state-of-knowledge of the reaction mechanisms and structures of extensively investigated FDMOs. The structural and functional properties, particularly common or unique features governing the versatility of the C4a-(hydro)peroxyflavin, are identified and used for explaining how the same common flavin intermediate can be used to manifest diverse reactions such as aromatic hydroxylation, N-hydroxylation, dehalogenation, halogenation, and light emission. A deep understanding of how FDMOs fine-tune their reactivities should be valuable in the future for designing FDMOs through enzyme engineering and creating potential biocatalysts for various applications. FDMOs comprise a diverse group of enzymes with a wide variety of structures and functions. The overall folding of FDMOs can be categorized into three different folding types including a Rossmann fold, TIM-barrel fold, and acyl-CoA dehydrogenase fold (16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar, 20Toplak M. Matthews A. Teufel R. The devil is in the details: the chemical basis and mechanistic versatility of flavoprotein monooxygenases.Arch. Biochem. Biophys. 2021; 698108732Google Scholar). Despite the variations in their structures, FDMOs share a common trait—the utilization of a flavin cofactor for oxygen activation. These foldings provide structural arrangements that facilitate the recognition of flavin binding. FDMOs can utilize either FAD or FMN; their specificity is predominantly determined by two factors (1): spatial accommodation or the availability of space to accommodate the flavin cofactor and (2) binding site environment or the interactions within the binding site that enable the recognition of the ADP portion of the flavin molecule. Notably, this recognition pattern is a distinctive characteristic of enzymes with a Rossmann fold such as 3-hydroxy-benzoate 4-hydroxylase (PHBH) from Pseudomonas fluorescens and flavin-dependent halogenase (Thal) from Streptomyces albogriseolus (Fig. 2A) (16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar, 23Entsch B. Ballou D.P. Massey V. Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase.J. Biol. Chem. 1976; 251: 2550-2563Google Scholar, 24Phintha A. Prakinee K. Jaruwat A. Lawan N. Visitsatthawong S. Kantiwiriyawanitch C. et al.Dissecting the low catalytic capability of flavin-dependent halogenases.J. Biol. Chem. 2021; 296100068Google Scholar, 25Hanukoglu I. Proteopedia: Rossmann fold: a beta-alpha-beta fold at dinucleotide binding sites.Biochem. Mol. Biol. Educ. 2015; 43: 206-209Google Scholar). The shared features among FDMOs recognizing ADP include interactions between polar and/or charged residues and the pyrophosphate and/or ribose sugar moieties of ADP (Fig. 2A). This interaction is particularly prominent in enzymes having the Rossmann fold mentioned above. Interestingly, for FDMOs which possess the acyl-CoA dehydrogenase fold instead of the Rossmann fold for recognizing FAD—such as group D: 4-hydroxyphenylacetate (HPA) 3-monooxygenase from Escherichia coli (HpaB) (26Deng Y. Faivre B. Back O. Lombard M. Pecqueur L. Fontecave M. Structural and functional characterization of 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli.ChemBioChem. 2020; 21: 163-170Google Scholar) and flavin-dependent dehalogenase (HadA) from Ralstonia pickettii DTP0602 (26Deng Y. Faivre B. Back O. Lombard M. Pecqueur L. Fontecave M. Structural and functional characterization of 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli.ChemBioChem. 2020; 21: 163-170Google Scholar)—these enzymes also possess similar interaction features as those with the Rossman fold within a segment designated as the "flavin binding loop" (Fig. 2B). This loop aids in the recognition of ADP, enabling these enzymes to bind specifically to FAD or FADH-. Conversely, enzymes having the TIM-barrel fold (e.g., groups C and H of FDMOs) contain binding sites where the isoalloxazine ring binds deeply within the protein structure such as bacterial luciferase (LuxAB) (Fig. 2C). Because this binding configuration lacks the necessary space to accommodate ADP binding, these FDMOs only bind to FMNH- as their substrate. An intriguing exception is observed in some cases of group D enzymes such as 3-HPA 4-hydroxylase from Acinetobacter baumannii (HPAH, the oxygenase component of HPAH or C2) and 4-HPA 3-monooxygenase (TtHpaB) from Thermus thermophilus HB8, in which a pocket for the isoalloxazine ring is situated near the protein surface (Fig. 2D) (15Chenprakhon P. Wongnate T. Chaiyen P. Monooxygenation of aromatic compounds by flavin-dependent monooxygenases.Protein Sci. 2019; 28: 8-29Google Scholar). This structural arrangement allows the side chain of flavin cofactors to extend outward from the enzyme structure, enabling both FADH- and FMNH- to be utilized as substrates. The flavin-binding site within FDMOs typically comprises of a spacious pocket capable of accommodating a flavin cofactor. This pocket exhibits a combination of hydrophobic and polar regions (Fig. 2A). The hydrophobic characteristics of this pocket create an optimal environment for accommodating the three aromatic rings of the isoalloxazine ring. Simultaneously, the polar regions are finely tuned to interact with specific moieties of the isoalloxazine ring around the N1, N3, C2-carbonyl oxygen, and C4-carbonyl oxygen (Fig. 2A). These distinctive environments are organized by the side chains and/or amino acid residue backbones. The substrate-binding domain within FDMOs exhibits remarkable diversity, enabling FDMOs to proficiently catalyze a wide spectrum of reactions (16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar, 20Toplak M. Matthews A. Teufel R. The devil is in the details: the chemical basis and mechanistic versatility of flavoprotein monooxygenases.Arch. Biochem. Biophys. 2021; 698108732Google Scholar). Despite the differences in the overall protein folding and variations in amino acid sequence, FDMOs maintain a consistent arrangement of substrate and flavin binding, positioning the substrate above the oxygen reacting site around the C4a position of the isoalloxazine ring on the re-side. This area is proposed to be the site where the reaction of reduced flavin and oxygen takes place, thus facilitating the generation of the C4a-(hydro)peroxyflavin intermediate (Fig. 2, E–H) (explained more in detail in the next section) (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 7Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37: 373-380Google Scholar). This mode of substrate binding is commonly observed in all FDMOs in which the substrate oxygenation occurs via a terminal oxygen atom transfer from C4a-(hydro)peroxyflavin onto a substrate. This binding arrangement appears across a range of substrates with diverse properties, including size, polarity, and steric characteristics. Examples include binding sites of halide ions in tryptophan halogenases, aromatic substrates in hydroxylases and dehalogenases, as well as cyclohexyl ketone in cyclohexanone monooxygenase (CHMO) (Fig. 2, E–H) (27Blasiak L.C. Drennan C.L. Structural perspective on enzymatic halogenation.Acc. Chem. Res. 2009; 42: 147-155Google Scholar, 28Agarwal V. Miles Z.D. Winter J.M. Eustáquio A.S. El Gamal A.A. Moore B.S. Enzymatic halogenation and dehalogenation reactions: pervasive and mechanistically diverse.Chem. Rev. 2017; 117: 5619-5674Google Scholar, 29Yachnin B.J. Sprules T. McEvoy M.B. Lau P.C.K. Berghuis A.M. The substrate-bound crystal structure of a baeyer–villiger monooxygenase exhibits a criegee-like conformation.J. Am. Chem. Soc. 2012; 134: 7788-7795Google Scholar, 30Alfieri A. Fersini F. Ruangchan N. Prongjit M. Chaiyen P. Mattevi A. Structure of the monooxygenase component of a two-component flavoprotein monooxygenase.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1177-1182Google Scholar, 31Pimviriyakul P. Jaruwat A. Chitnumsub P. Chaiyen P. Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking.J. Biol. Chem. 2021; 297100952Google Scholar). This binding arrangement likely facilitates robust interactions between substrate and C4a-(hydro)peroxyflavin, thus enabling convenient oxygen atom transfer (7Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37: 373-380Google Scholar). It should be noted that the mode of substrate binding at the re-side in C4a-(hydro)peroxyflavin-forming enzymes is not observed in FDMOs that form flavin N5-oxide. In the case of FDMOs that form flavin N5-oxide, the substrate was found at the si-face of the flavin, while the molecular oxygen is proposed to approach at the re-side (32Matthews A. Saleem-Batcha R. Sanders J.N. Stull F. Houk K.N. Teufel R. Aminoperoxide adducts expand the catalytic repertoire of flavin monooxygenases.Nat. Chem. Biol. 2020; 16: 556-563Google Scholar). This was explained by results from computational calculations which demonstrated that the mechanism of substrate oxygenation by flavin N5-oxide is different from C4a-(hydro)peroxyflavin in which flavin N5-oxide at the re-side is brought closer to the substrate at the si-face via N5-inversion (32Matthews A. Saleem-Batcha R. Sanders J.N. Stull F. Houk K.N. Teufel R. Aminoperoxide adducts expand the catalytic repertoire of flavin monooxygenases.Nat. Chem. Biol. 2020; 16: 556-563Google Scholar). With greater availability of structural and mechanistic information regarding FDMOs, it would be interesting to conduct comparative investigations across a broader range of FDMOs with diverse reactions and substrate utilizations. The analysis may provide a deeper understanding of the controlling features of the enzyme and lead to the ability to modify specific properties of FDMOs. As described above, FDMOs exhibit a unique structural arrangement, with the isoalloxazine ring of flavin positioned deep into the structure (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar). A fundamental question arises: how does the buried flavin cofactor that is secluded from the solvent environment react with oxygen to form the reactive C4a-(hydro)peroxyflavin intermediate? Extensive investigations into the reaction mechanism of reduced flavin with molecular oxygen have shed light on this process. It has been proposed that molecular oxygen diffuses through multiple tunnels, reaching the oxygen-binding pocket located at the re-side of the isoalloxazine ring, near the C4a position (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 7Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37: 373-380Google Scholar). This unique arrangement allows efficient formation of the C4a-(hydro)peroxyflavin intermediate, shields the intermediate from the surrounding solvent environment, and facilitates its prompt oxygen transfer reaction with the substrate (1Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118: 1742-1769Google Scholar, 16Paul C.E. Eggerichs D. Westphal A.H. Tischler D. van Berkel W.J.H. Flavoprotein monooxygenases: versatile biocatalysts.Biotechnol. Adv. 2021; 51107712Google Scholar). Recent crystal structures of FDMOs including a stabilizing flavin N5-oxide monooxygenase involved in enterocin biosynthesis (flavin N5-oxide stabilizing enterocin biosynthesis monooxygenase) from Streptomyces maritimus (33Saleem-Batcha R. Stull F. Sanders J.N. Moore B.S. Palfey B.A. Houk K.N. et al.Enzymatic control of dioxygen binding and functionalization of the flavin cofactor.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4909-4914Google Scholar) and a pyrimidine monooxygenase (RutA) from E. coli (32Matthews A. Saleem-Batcha R. Sanders J.N. Stull F. Houk K.N. Teufel R. Aminoperoxide adducts expand the catalytic repertoire of flavin monooxygenases.Nat. Chem. Biol. 2020; 16: 556-563Google Scholar) have identified the oxygen-binding location at the re-side of the flavin (Fig. 2I). The data are also valuable in confirming the oxygen reaction site for other FDMOs such as HPAH and HadA (Fig. 2J) in which their oxygen reaction site have been identified by computational calculations (30Alfieri A. Fersini F. Ruangchan N. Prongjit M. Chaiyen P. Mattevi A. Structure of the monooxygenase component of a two-component flavoprotein monooxygenase.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1177-1182Google Scholar, 31Pimviriyakul P. Jaruwat A. Chitnumsub P. Chaiyen P. Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking.J. Biol. Chem. 2021; 297100952Google Scholar, 34Visitsatthawong S. Chenprakhon P. Chaiyen P. Surawatanawong P. Mechanism of oxygen activation in a flavin-dependent monooxygenase: a nearly barrierless formation of C4a-hydroperoxyflavin via proton-coupled electron transfer.J. Am. Chem. Soc. 2015; 137: 9363-9374Google Scholar). These data significantly advance our understanding of the oxygen activation reaction by FDMOs and demonstrate how these enzymes effectively control the reaction of reduced flavin and oxygen in order to generate the critical C4a-(hydro)peroxyflavin intermediate essential for substrate functionalization. A distinctive feature of FDMOs is their ability to generate C4a-(hydro)peroxyflavin as an intermediate during their catalytic reactions. The stability of C4a-(hydro)peroxyflavin varies among the different enzymes. In most cases, this intermediate can only be detected using rapid kinetic studies (35Yeh E. Blasiak L.C. Koglin A. Drennan C.L. Walsh C.T. Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases.Biochemistry. 2007; 46: 1284-1292Google Scholar, 36Ruangchan N. Tongsook C. Sucharitakul J. Chaiyen P. pH-dependent studies reveal an efficient hydroxylation mechanism of the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase.J. Biol. Chem. 2011; 286: 223-233Google Scholar). The ability of FDMOs to stabilize C4a-(hydro)peroxyflavin is among the key distinctions of FDMOs from flavoenzyme oxidases in which the intermediate could not be detected during their catalytic cycles, either because it is not formed or because it rapidly decays (37Massey V. Activation of molecular oxygen by flavins and flavoproteins.J. Biol. Chem. 1994; 269: 22459-22462Google Scholar, 38Mattevi A. To be or not to be an oxidase: challenging the oxygen reactivity of flavoenzymes.Trends Biochem. Sci. 2006; 31: 276-283Google Scholar, 39Chakraborty S. Ortiz-Maldonado M. Entsch B. Ballou D.P. Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase.Biochemistry. 2010; 49: 372-385Google Scholar). The exceptions were found for the reactions of pyranose 2-oxidase from Trametes multicolor, where the C4a-hydroperoxyflavin was detected during the oxidative half-reaction, and choline oxidase in which the flavin C4a-adduct could be detected by single-crystal spectroscopic method and protein crystallization (40Sucharitakul J. Prongjit M. Haltrich D. Chaiyen P. Detection of a C4a-hydroperoxyflavin intermediate in the reaction of a flavoprotein oxidase.Biochemistry. 2008; 47: 8485-8490Google Scholar, 41Orville A.M. Lountos G.T. Finnegan S. Gadda G. Prabhakar R. Crystallographic, spectroscopic, and computational analysis of a flavin C4a−Oxygen adduct in choline oxidase.Biochemistry. 2009; 48: 720-728Google Scholar). Comparison of the structures of FDMOs and oxidases is instrumental for identifying the critical factors within the C4a-N5 locus that stabilize the C4a-(hydro)peroxyflavin intermediate. Site-directed mutagenesis of HPAH demonstrated the significance of Ser171, located in close proximity to, and forming an H-bond with the N5, for stabilization of the C4a-hydroperoxyflavin intermediate (Fig. 2K). Replacing this residue with Ala resulted in a dramatic decrease in intermediate stabilization (42Thotsaporn K. Chenprakhon P. Sucharitakul J. Mattevi A. Chaiyen P. Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms.J. Biol. Chem. 2011; 286: 28170-28180Google Scholar). Similar interactions were also found in the Thr residues of TtHpaB (43Kim S.H. Hisano T. Takeda K. Iwasaki W. Ebihara A. Miki K. Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.J. Biol. Chem. 2007; 282: 33107-33117Google Scholar), chlorophenol 4-monooxygenase (TftD) from Burkholderia cepacia AC1100 (44Webb B.N. Ballinger J.W. Kim E. Belchik S.M. Lam K.-S. Youn B. et al.Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:FAD oxidoreductase (TftC) of Burkholderia cepacia AC1100.J. Biol. Chem. 2010; 285: 2014-2027Google Scholar), and HadA (31Pimviriyakul P. Jaruwat A. Chitnumsub P. Chaiyen P. Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking.J. Biol. Chem. 2021; 297100952Google Scholar). As these enzymes are those which can stabilize C4a-(hydro)peroxyflavin well during their catalytic cycles (7Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37: 373-380Google Scholar, 45Pimviriyakul P. Thotsaporn K. Sucharitakul J. Chaiyen P. Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA.J. Biol. Chem. 2017; 292: 4818-4832Google Scholar), the H-bonding interaction around flavin N5 is thus thought to be important for C4a-(hydro)peroxyflavin stabilization. Interestingly, in the case of Baeyer–Villiger monooxygenases (BVMOs) and N-hydroxylating monooxygenases (NMOs), despite the absence of an amino acid residue capable of forming a hydrogen bond with the N5, the binding of NADP+ is required for stabilization of C4a-(hydro)peroxyflavin (for periods of up to hours, that is, in the reactions of CHMO from Acinetobacter sp. NCIMB 9871 (46Sheng D. Ballou D.P. Massey V. Mechanistic studies of cyclohexanone monooxy