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Flavin-containing monooxygenase from Methylophaga sp. ( m FMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored m FMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis ( Ni FMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of m FMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o -hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o -position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o -hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o -hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.

This short review reports the surprising phenomenon of nuclear hyperpolarization occurring in chemical reactions, which is called CIDNP (chemically induced dynamic nuclear polarization) or photo-CIDNP if the chemical reaction is light-driven. The phenomenon occurs in both liquid and solid-state, and electron transfer systems, often carrying flavins as electron acceptors, are involved. Here, we explain the physical and chemical properties of flavins, their occurrence in spin-correlated radical pairs (SCRP) and the possible involvement of flavin-carrying SCRPs in animal magneto-reception at earth's magnetic field.

Flavins are a class of organic compounds with the basic structure of 7,8-dimethy-10-alkyl isoalloxazine. They are ubiquitous in nature and participate in many biochemical reactions. Due to various existing forms, there is a lack of systematic research on the absorption and fluorescence spectra of flavins. In this study, employing the density functional theory (DFT) and time-dependent (TD) DFT, we calculated the pH-dependent absorption and fluorescence spectra of flavin of three redox states (quinone, semiquinone, and hydroquinone) in solvents. The chemical equilibrium of three redox states of flavins and the pH effect on the absorption spectra and fluorescence spectra of flavins were carefully discussed. The conclusion helps with identifying the existing forms of flavins in solvent with different pH values.

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re -side and si -side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pK a1 = 6.6 and pK a2 = 9.2 at 2 μM FMN while it contained only a descending limb pK a of 9.7 at 50 μM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS -hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin.

Natural products are usually highly complicated organic molecules with special scaffolds, and they are an important resource in medicine. Natural products with complicated structures are produced by enzymes, and this is still a challenging research field, its mechanisms requiring detailed methods for elucidation. Flavin adenine dinucleotide (FAD)-dependent monooxygenases (FMOs) catalyze many oxidation reactions with chemo-, regio-, and stereo-selectivity, and they are involved in the synthesis of many natural products. In this review, we introduce the mechanisms for different FMOs, with the classical FAD (C4a)-hydroperoxide as the major oxidant. We also summarize the difference between FMOs and cytochrome P450 (CYP450) monooxygenases emphasizing the advantages of FMOs and their specificity for substrates. Finally, we present examples of FMO-catalyzed synthesis of natural products. Based on these explanations, this review will expand our knowledge of FMOs as powerful enzymes, as well as implementation of the FMOs as effective tools for biosynthesis.

Protein tyrosine kinases (PTKs) are the most potential therapeutic targets for cancer. Herein, we present a sound rationale for synthesis of a series of novel 2-(methylthio), 2-(substituted alkylamino), 2-(heterocyclic substituted), 2-amino, 2,4-dioxo and 2-deoxo-5-deazaalloxazine derivatives by applying structure-based drug design (SBDD) using AutoDock 4.2. Their antitumor activities against human CCRF-HSB-2, KB, MCF-7 and HeLa have been investigated in vitro. Many 5-deazaalloxazine analogs revealed high selective activities against MCF-7 tumor cell lines (IC 50 : 0.17-2.17 µM) over HeLa tumor cell lines (IC 50 > 100 µM). Protein kinase profiling revealed that compound 3h induced multi- targets kinase inhibition including -43% against (FAK), -40% against (CDKI) and -36% against (SCR). Moreover, the Annexin-V/PI apoptotic assay elucidate that compound 3h showed 33% and potentially 140% increase in early and late apoptosis to MCF-7 cells respectively, compared to the control. The structure-activity relationship (SAR) and molecular docking study using PTK as a target enzyme for the synthesized 7-deazaalloaxazine derivatives were investigated as potential antitumor agents. The AutoDock binding affinities of the 5-deazaalloxazine analogs into c-kit PTK (PDB code: 1t46) revealed reasonable correlations between their AutoDock binding free energy and IC 50 ., Competing Interests: Authors declared that there is no actual or potential conflict of interest and have approved the article.

In this review, recent progress in the engineering of the oxidative half-reaction of flavin-dependent oxidases and dehydrogenases is discussed, considering their current and future applications in bioelectrochemical studies, such as for the development of biosensors and biofuel cells. There have been two approaches in the studies of oxidative half-reaction: engineering of the oxidative half-reaction with oxygen, and engineering of the preference for artificial electron acceptors. The challenges for engineering oxidative half-reactions with oxygen are further categorized into the following approaches: (1) mutation to the putative residues that compose the cavity where oxygen may be located, (2) investigation of the vicinities where the reaction with oxygen may take place, and (3) investigation of possible oxygen access routes to the isoalloxazine ring. Among these approaches, introducing a mutation at the oxygen access route to the isoalloxazine ring represents the most versatile and effective strategy. Studies to engineer the preference of artificial electron acceptors are categorized into three different approaches: (1) engineering of the charge at the residues around the substrate entrance, (2) engineering of a cavity in the vicinity of flavin, and (3) decreasing the glycosylation degree of enzymes. Among these approaches, altering the charge in the vicinity where the electron acceptor may be accessed will be most relevant., Competing Interests: The authors declare no conflict of interest.

Halogens can be very important for active agents as vital parts of their binding mode, on the one hand, but are on the other hand instrumental in the synthesis of most active agents. However, the primary halogenating compound is molecular chlorine which has two major drawbacks, high energy consumption and hazardous handling. Nature bypassed molecular halogens and evolved at least six halogenating enzymes: Three kind of haloperoxidases, flavin-dependent halogenases as well as α-ketoglutarate and S- adenosylmethionine (SAM)-dependent halogenases. This review shows what is known today on these enzymes in terms of biocatalytic usage. The reader may understand this review as a plea for the usage of halogenating enzymes for fine chemical syntheses, but there are many steps to take until halogenating enzymes are reliable, flexible, and sustainable catalysts for halogenation.

Previously unreported meroterpene, acremine S ( 1 ), and benzopyran derivative, acremine T ( 2 ), were isolated, together with lumichrome ( 3 ), ergosterol ( 4 ) and ergosterol 5,8-endoperoxide, from cultures of the marine sponge-associated fungus Acremonium persicinum KUF1007. The structure of the previously unreported compounds was established based on an extensive analysis of 1D and 2D NMR spectra as well as HRMS data. The absolute configurations of the stereogenic centers of 1 were established, unambiguously, based on NOESY correlations and comparison of calculated and experimental electronic circular dichroism (ECD) spectra. Compounds 1-3 were tested for their in vitro acetylcholinesterase and butyrylcholinesterase inhibitory activities.

Flavins are known to be extremely versatile, thus enabling routes to innumerable modifications in order to obtain desired properties. Thus, in the present paper, the group of bio-inspired conjugated materials based on the alloxazine core is synthetized using two efficient novel synthetic approaches providing relatively high reaction yields. The comprehensive characterization of the materials, in order to evaluate the properties and application potential, has shown that the modification of the initial alloxazine core with aromatic substituents allows fine tuning of the optical bandgap, position of electronic orbitals, absorption and emission properties. Interestingly, the compounds possess multichromophoric behavior, which is assumed to be the results of an intramolecular proton transfer.

Flavin chromophores play key roles in a wide range of photoactive proteins, but key questions exist in relation to their fundamental spectroscopic and photochemical properties. In this work, we report the first gas-phase spectroscopy study of protonated alloxazine (AL∙H⁺), a model flavin chromophore. Laser photodissociation is employed across a wide range (2.34⁻5.64 eV) to obtain the electronic spectrum and characterize the photofragmentation pathways. By comparison to TDDFT quantum chemical calculations, the spectrum is assigned to two AL∙H⁺ protomers; an N5 (dominant) and O4 (minor) form. The protomers have distinctly different spectral profiles in the region above 4.8 eV due to the presence of a strong electronic transition for the O4 protomer corresponding to an electron-density shift from the benzene to uracil moiety. AL∙H⁺ photoexcitation leads to fragmentation via loss of HCN and HNCO (along with small molecules such as CO₂ and H₂O), but the photofragmentation patterns differ dramatically from those observed upon collision excitation of the ground electronic state. This reveals that fragmentation is occurring during the excited state lifetime. Finally, our results show that the N5 protomer is associated primarily with HNCO loss while the O4 protomer is associated with HCN loss, indicating that the ring-opening dynamics are dependent on the location of protonation in the ground-state molecule.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid., Competing Interests: The authors declare no conflict of interest.

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O₂-dependent oxidation of prim -alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to "over-oxidation" of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis-Menten kinetics were performed to corroborate these findings., Competing Interests: The authors declare no conflict of interest.

In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

A click chemistry approach based on the reaction between alkynylflavins and mono(6-azido-6-deoxy)-β-cyclodextrin has proven to be a useful tool for the synthesis of flavin-cyclodextrin conjugates studied as monooxygenase mimics in enantioselective sulfoxidations.

In a previous study, we showed that formylmethylflavin (FMF) can bind to cysteine. In this study, FMF was reacted with native peptides (CG and CKLVFF) containing an N-terminal cysteine. The formation of flavin-CG and flavin-CKLVFF was confirmed using HPLC and ESI-MS. Storage of flavin-CKLVFF in DMSO at -30 °C for 7 days resulted in no detectable deposition. In contrast, flavin-CKLVFF formed deposits when stored in water at -30 °C for 1 day, but no deposit was observed in the aqueous solution of flavin-CKLVFF after 7 days storage in the presence of 0.1% Triton X-100.

Two acceptor-acceptor dyads have been synthesized featuring a flavin moiety and a naphthalenediimide (NDI) unit. The NDI unit is linked to the flavin through a short spacer group via either the N(3) or N(10) positions of the flavin. We have investigated the UV-Vis and redox properties of these multi-electron accepting systems which indicate that these materials display the collective properties of their component systems. Fluorescence spectroscopy measurements have revealed that their emission properties are dominated by the flavin unit.

Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a "molecular scaffold" in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates.

The blue-light photo-reduction (conversion of oxidized flavin quinone via flavin semiquinone to fully reduced flavin hydroquinone) and dark re-oxidation of the flavins riboflavin and lumiflavin in starch (α-amylose) films was studied by absorption and luminescence spectroscopy. Blue-light photo-excitation caused an absorption, fluorescence, and phosphorescence decrease which recovered in the dark. The photo-reduction dark-oxidation cycle could be repeated. The efficiency of photo-reduction decreased with exposed excitation energy, and the speed of re-oxidation in the dark slowed down with time after excitation. The absorption did not fully recover. The fluorescence efficiency after a long time of storage in the dark increased beyond the initial flavin quinone fluorescence efficiency. Flavin photo-excitation is thought to cause starch-flavin restructuring (static fluorescence quenching center formation), enabling enhanced photo-induced starch to flavin electron transfer with subsequent flavin reduction and starch oxidation. In the dark, after light switch-off, thermal reversion of flavin reduction and starch oxidation occurred.

A series of pyrimido[4,5-b]quinolines (5-deazaflavines), were synthesized by microwave assisted intramolecular cyclization. The N⁴-substituted-2,4-diamino-6-chloro-pyrimidine-5-carbaldehydes, were prepared by selective monoamination of 2-amino-4,6-dichloropyrimidine-5-carbaldehyde with aliphatic and aromatic amines.