From Ground-State to Excited-State Activation Modes: Flavin-Dependent "Ene"-Reductases Catalyzed Non-natural Radical Reactions.

ConspectusEnzymes are desired catalysts for chemical synthesis, because they can be engineered to provide unparalleled levels of efficiency and selectivity. Yet, despite the astonishing array of reactions catalyzed by natural enzymes, many reactivity patterns found in small molecule catalysts have no counterpart in the living world. With a detailed understanding of the mechanisms utilized by small molecule catalysts, we can identify existing enzymes with the potential to catalyze reactions that are currently unknown in nature. Over the past eight years, our group has demonstrated that flavin-dependent "ene"-reductases (EREDs) can catalyze various radical-mediated reactions with unparalleled levels of selectivity, solving long-standing challenges in asymmetric synthesis.This Account presents our development of EREDs as general catalysts for asymmetric radical reactions. While we have developed multiple mechanisms for generating radicals within protein active sites, this account will focus on examples where flavin mononucleotide hydroquinone (FMNhq) serves as an electron transfer radical initiator. While our initial mechanistic hypotheses were rooted in electron-transfer-based radical initiation mechanisms commonly used by synthetic organic chemists, we ultimately uncovered emergent mechanisms of radical initiation that are unique to the protein active site. We will begin by covering intramolecular reactions and discussing how the protein activates the substrate for reduction by altering the redox-potential of alkyl halides and templating the charge transfer complex between the substrate and flavin-cofactor. Protein engineering has been used to modify the fundamental photophysics of these reactions, highlighting the opportunity to tune these systems further by using directed evolution. This section highlights the range of coupling partners and radical termination mechanisms available to intramolecular reactions.The next section will focus on intermolecular reactions and the role of enzyme-templated ternary charge transfer complexes among the cofactor, alkyl halide, and coupling partner in gating electron transfer to ensure that it only occurs when both substrates are bound within the protein active site. We will highlight the synthetic applications available to this activation mode, including olefin hydroalkylation, carbohydroxylation, arene functionalization, and nitronate alkylation. This section also discusses how the protein can favor mechanistic steps that are elusive in solution for the asymmetric reductive coupling of alkyl halides and nitroalkanes. We are aware of several recent EREDs-catalyzed photoenzymatic transformations from other groups. We will discuss results from these papers in the context of understanding the nuances of radical initiation with various substrates.These biocatalytic asymmetric radical reactions often complement the state-of-the-art small-molecule-catalyzed reactions, making EREDs a valuable addition to a chemist's synthetic toolbox. Moreover, the underlying principles studied with these systems are potentially operative with other cofactor-dependent proteins, opening the door to different types of enzyme-catalyzed radical reactions. We anticipate that this Account will serve as a guide and inspire broad interest in repurposing existing enzymes to access new transformations.

Cyclodipeptide oxidase is an enzyme filament.

Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,ß-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.

Flavin mononucleotide regulated photochemical isomerization and degradation of zeatin.

cis-Zeatin (cZ), a cytokinin often overlooked compared to trans-zeatin (tZ), can now be controlled in live cells and plants through a new biocompatible reaction. Using flavin photosensitizers, cZ can be isomerized to tZ or degraded, depending on the presence of a reducing reagent. This breakthrough offers a novel approach for regulating plant growth through chemical molecules.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.

Cloning and characterization of FMN-dependent azoreductases from textile industry effluent identified through metagenomic sequencing.

Azo dyes, when released untreated in the environment, cause detrimental effects on flora and fauna. Azoreductases are enzymes capable of cleaving commercially used azo dyes, sometimes in less toxic by-products which can be further degraded via synergistic microbial cometabolism. In this study, azoreductases encoded by FMN1 and FMN2 genes were screened from metagenome shotgun sequences generated from the samples of textile dye industries' effluents, cloned, expressed, and evaluated for their azo dye decolorization efficacy. At pH 7 and 45°C temperature, both recombinant enzymes FMN1 and FMN2 were able to decolorize methyl red at 20 and 100 ppm concentrations, respectively. FMN2 was found to be more efficient in decolorization/degradation of methyl red than FMN1. This study offers valuable insights into the possible application of azoreductases to reduce the environmental damage caused by azo dyes, with the hope of contributing to sustainable and eco-friendly practices for the environment management. This enzymatic approach offers a promising solution for the bioremediation of textile industrial effluents. However, the study acknowledges the need for further process optimization to enhance the efficacy of these enzymes in large-scale applications.Implications: The study underscores the environmental hazards associated with untreated release of azo dyes into the environment and emphasizes the potential of azoreductases, specifically those encoded by FMN1 and FMN2 genes, to mitigate the detrimental effects. The study emphasizes the ongoing commitment to refining and advancing the enzymatic approach for the bioremediation of azo dye-containing effluents, marking a positive stride toward more sustainable industrial practices.

(De)carboxylation mechanisms of heteroaromatic substrates catalyzed by prenylated FMN-dependent UbiD decarboxylases: An in-silico study.

The UbiD enzymes are proposed to catalyze reversible (de)carboxylation reaction of unsaturated carboxylic acids using prenylated flavin mononucleotide (prFMN) as a cofactor. This positions UbiD enzymes as promising candidates for converting CO2 into valuable chemicals. However, their industrial-scale biotransformation is currently constrained by low conversion rates attributed to thermodynamic limitations. To enhance the carboxylation activity of UbiD enzymes, a molecular-level understanding of the (de)carboxylation mechanisms is necessary. In this study, we investigated the reaction mechanisms of heteroaromatic substrates catalyzed by PtHmfF, PaHudA, and AnlnD enzymes using molecular dynamics (MD) simulations and free energy calculations. Our extensive mechanistic study elucidates the mechanisms involved in the formation of the initial prFMN-substrate intermediate. Specifically, we observed nucleophilic attack during decarboxylation, while carboxylation reactions involving furoic acid, pyrrole, and indole tend to favor a 1,3-dipolar cycloaddition mechanism. Furthermore, we identified proton transfer as the rate-limiting step in the carboxylation reaction. In addition, we considered the perspectives of reaction energies and electron transfer to understand the distinct mechanisms underlying decarboxylation and carboxylation. Our calculated free energies are consistent with available experimental kinetics data. Finally, we explored how different rotamers of catalytic residues influence the efficiency of the initial intermediate formation.

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production.

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03-2.42 and 3.83-4.94 folds, after exposure to 50 and 200 µM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03-31.71%, 22.86-26.04%, and 28.51-33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Effect of Fermentation Scale on Microbiota Dynamics and Metabolic Functions for Indigo Reduction.

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

Backbone Cyclization of Flavin Mononucleotide-Based Fluorescent Protein Increases Fluorescence and Stability.

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

Flavin mononucleotide in visible light photoinitiating systems for multiple-photocrosslinking and photoencapsulation strategies.

Visible light-induced photocrosslinking techniques have attracted significant attention for their flexibility, controllability, safety, and energy conservation, especially in tissue engineering and biofabrication, compared to UV photocrosslinking. Despite these advantages, current photoinitiators are constrained by various challenges, including inadequate photoinitiation efficiency, low biocompatibility, poor water solubility, and limited compatibility with diverse crosslinking systems. Here, a water-soluble derivative of riboflavin, flavin mononucleotide (FMN-), was used to assess its potential as an initiator in multiple-photocrosslinking systems, including radical photopolymerization, dityrosine, and ditryptophan coupling crosslinking, under blue light irradiation. Blue light irradiation facilitated an efficient electron transfer reaction between FMN- and persulfate, owing to their suitable spectral compatibility and photoactivity. The resulting oxidizing free radicals and excited triplet state of FMN- served as initiating active species for the multiple-photocrosslinking reactions. The combination of FMN- and potassium persulfate (KPS) exhibited exceptional photoinitiation efficiency for various biomaterials, including silk fibroin, gelatin, poly(ethylene glycol) diacrylate, and carboxymethyl cellulose modified with amino acids. Furthermore, the cytocompatibility of the FMN-/KPS photoinitiator was demonstrated by the survival rates of 3T3-LI fibroblasts encapsulated in it, which exceeded 95 % when compared to a commercial initiator. STATEMENT OF SIGNIFICANCE: By introducing persulfate, the photoinitiation efficiency of flavin mononucleotide was significantly improved. The application scenarios of flavin mononucleotide and persulfate combinations were also greatly extended, including radical photopolymerization, dityrosine, diphenylalanine, and ditryptophan coupling crosslinking. Among them, the coupling crosslinking of amino acids (di-phenylalanine, and di-tryptophan) modified carboxymethyl cellulose, to our knowledge, was first reported. The excellent cytocompatibility of cell encapsulation further proved that the combinations of flavin mononucleotide and persulfate have great potential in tissue engineering.

Medicinal benefits, biological, and nanoencapsulation functions of riboflavin with its toxicity profile: A narrative review.

Riboflavin is a precursor of the essential coenzymes flavin mononucleotide and flavin adenine dinucleotide. Both possess antioxidant properties and are involved in oxidation-reduction reactions, which have a significant impact on energy metabolism. Also, the coenzymes participate in metabolism of pyridoxine, niacin, folate, and iron. Humans must obtain riboflavin through their daily diet because of the lack of programmed enzymatic machineries for de novo riboflavin synthesis. Because of its physiological nature and fast elimination from the human body when in excess, riboflavin consumed is unlikely to induce any negative effects or develop toxicity in humans. The use of riboflavin in pharmaceutical and clinical contexts has been previously explored, including for preventing and treating oxidative stress and reperfusion oxidative damage, creating synergistic compounds to mitigate colorectal cancer, modulating blood pressure, improving diabetes mellitus comorbidities, as well as neuroprotective agents and potent photosensitizer in killing bloodborne pathogens. Thus, the goal of this review is to provide a comprehensive understanding of riboflavin's biological applications in medicine, key considerations of riboflavin safety and toxicity, and a brief overview on the nanoencapsulation of riboflavin for various functions including the treatment of a range of diseases, photodynamic therapy, and cellular imaging.

Enzymes in riboflavin biosynthesis: Potential antibiotic drug targets.

The rapid resistance of pathogens to antibiotics has emerged as a major threat to global health. Identification of new antibiotic targets is thus needed for developing alternative drugs. Genes encoding enzymes involved in the biosynthesis of riboflavin and flavin cofactors (FMN/FAD) are attractive targets because these enzymatic reactions are necessary for most bacteria to synthesize flavin cofactors for use in their central metabolic reactions. Moreover, humans lack most of these enzymes because we uptake riboflavin from our diet. This review discusses the current knowledge of enzymes involved in bacterial biosynthesis of riboflavin and other flavin cofactors, as well as the functions of the FMN riboswitch. Here, we highlight recent progress in the structural and mechanistic characterization, and inhibition of GTP cyclohydrolase II (GCH II), lumazine synthase (LS), riboflavin synthase (RFS), FAD synthetase (FADS), and FMN riboswitch, which have been identified as plausible antibiotic targets. As the structures and functions of these enzymes and regulatory systems are not completely understood, they are attractive as subjects for future in-depth biochemical and biophysical analysis.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

Reaction mechanism and kinetics of the two-component flavoprotein dimethyl sulfone monooxygenase system: Using hydrogen peroxide for monooxygenation and substrate cleavage.

The dimethyl sulfone monooxygenase system is a two-component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO2 ) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor, catalyzing flavin reduction using NADH. The monooxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH- with a Kd of 17.4 ± 0.9 µm and 4.08 ± 0.8 µm, respectively. The binding of FMN to DMSMO is required prior to binding DMSO2 . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO2 . Substituting reduced DMSR with FMNH- demonstrated the same oxidation kinetics, indicating that FMNH- from DMSR was transferred to DMSMO. The oxidation of FMNH- :DMSMO, with and without DMSO2 did not generate any flavin adducts for monooxygenation. Therefore, H2 O2 is likely to be the reactive agent to attack the substrate. The H2 O2 assay results demonstrated production of H2 O2 from the oxidation of FMNH- :DMSMO, whereas H2 O2 was not detected in the presence of DMSO2 , confirming H2 O2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H2 O2 rapidly reacts with DMSO2 , with flavin oxidation as the rate-limiting step. This is the first report of the kinetic mechanisms of both components using rapid kinetics and of a method for methanesulfinate detection using LC-MS.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Insight into the enhancing mechanism of exogenous electron mediators on biological denitrification in microbial electrolytic cell.

Sustained nitrate accumulation in surface water ecosystem was continuously grabbing public attention. Autotrophic denitrification by electron supplement has been applied to overcome the requirement of carbon source, thus the new problem that how to improve the efficiency of extracellular electrons transfer to denitrifiers comes to us. The addition of exogenous electron mediators has been considered as an important strategy to promote extracellular electrons transfer in reductive metabolism. To date, knowledge is lacking about the promoting effects and pathways in nitrate removal by electron mediators. Here, we fully investigated the performance of nitrogen removal as well as quantified the characteristics of biofilms with six electron mediators (riboflavin, flavin mononucleotide, AQS, AQDS, biochar and Nano-Fe3O4) treating in microbial electrolytic cell system. The six electron mediators promoted nitrate removal rate by 76.03-90.43 % with electron supplement. The growth and activity of cathodic biofilm, conductive nanowires generation and electrochemically active substance synthesis of extracellular polymeric substances were facilitated by electron mediator addition. Electrochemical analysis revealed that conductivity and redox capacity of cathodic biofilm was increased for accelerating electron transfer. Moreover, they upregulated the abundance of denitrifying communities and denitrifying genes accordingly. Their denitrification efficiency varied due to their promotion ability in the above different strategies and conductive characteristics, and the efficiency could be concluded as: Nano-Fe3O4 > riboflavin > flavin mononucleotide > AQS ≈ AQDS > biochar. This study revealed how addition of electron mediators promoted denitrification with electron supplement, and compared their promoting efficiency in several main aspects.

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors.

Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1-3 steps with moderate yields (10-57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.

QM/MM Modeling of the Flavin Functionalization in the RutA Monooxygenase.

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.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging.

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Naphthalene Carboxylation in the Sulfate-Reducing Enrichment Culture N47 Is Proposed to Proceed via 1,3-Dipolar Cycloaddition to the Cofactor Prenylated Flavin Mononucleotide.

Polycyclic aromatic hydrocarbons are persistent pollutants of anthropogenic or natural origin in the environment and accumulate in anoxic habitats. In this study, we investigated the mechanism of the enzyme naphthalene carboxylase as a model reaction for polycyclic aromatic hydrocarbon activation by carboxylation. An enzyme assay was established with cell extracts of the highly enriched culture N47. In assays without addition of ATP, naphthalene carboxylase catalyzed a stable isotope exchange of the carboxyl group of naphthoate with 13C-labeled bicarbonate buffer, which can only occur via a partial backwards reaction of the naphthalene carboxylase reaction to an intermediate that does not include the carboxyl group. Hence, a new carboxyl group from the labeled bicarbonate is added upon forward reaction to the naphthoate. This indicates that the reaction mechanism consists of two or more steps and that at least the latter steps are reversible and ATP independent. Naphthalene carboxylation assays were carried out in deuterated buffer and revealed the incorporation of 0, 1, 2, or 3 deuterium atoms in the final product naphthoyl-coenzyme A, indicating that the reaction is fully reversible. Putative reaction mechanisms were tested by quantum mechanical calculations. The proposed mechanism of the reaction consists of three steps: the activation of the naphthalene by 1,3-dipolar cycloaddition of the cofactor prFMN to naphthalene, release of a proton and rearomatization producing a stable intermediate, and a carboxylation with a reverse 1,3-dipolar cycloaddition and cleavage of the bond to the cofactor producing 2-naphthoate. IMPORTANCE Pollution with polycyclic aromatic hydrocarbons poses a great hazard to humans and animals, with considerable long-term effects. The anaerobic degradation of polycyclic aromatic hydrocarbons in anoxic zones and anaerobic growth of such organisms is very slow, leading to only poor investigation of the degradation pathways, so far. In this work, we elucidated the mechanism of naphthalene carboxylase, a key enzyme in anaerobic naphthalene degradation. This is the first mechanism proposed for a carboxylase targeting nonsubstituted (polycyclic) aromatic compounds and can serve as a model for the initial activation reaction in the anaerobic degradation of benzene or nonsubstituted polycyclic aromatic hydrocarbons, as well as similar enzymatic reactions from the expanding class of UbiD-like (de)carboxylases.