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Flavin‐dependent nitroreductases are gaining attention as biocatalysts for the synthesis of pharmaceutically active compounds and their precursors. Here, we examined a panel of nitroreductase‐like flavoenzymes for their reductase activity towards a wide variety of aromatic and aliphatic nitro compounds, nitroolefins, and α,β‐unsaturated carbonyl compounds. Several of these flavoenzymes displayed high reductase activity and achieved excellent conversion of diverse nitroarenes, nitroolefins and α,β‐unsaturated carbonyl compounds, accomplishing good product yields in semi‐preparative scale reactions (up to 97%). In addition to the catalytic promiscuity of several of these flavoenzymes, being able to perform the reduction of nitro groups (nitroreductase activity) as well as C=C groups (ene‐reductase activity), this study also revealed that some flavoenzymes exhibit high chemo‐, regio‐ and/or enantioselectivity, making them attractive enzymes for use in organic synthesis. [ABSTRACT FROM AUTHOR]

Genetically encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin two light-oxygen-voltage flavoprotein have been developed to photochemically generate singlet oxygen (¹O2) in vitro and in vivo, yet the effect of ¹O2 on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" light-oxygen-voltage flavoprotein is capable of photochemical ¹O2 generation. Once generated, ¹O2 induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein--protein cross-links may interfere with native biological responses to ¹O2, which can be ameliorated by substitution of the surface exposed histidines of improved" lightoxygen- voltage or other ¹O2-generating flavoproteins. [ABSTRACT FROM AUTHOR]

Background: Monoamine oxidases (MAO) are flavoenzymes that metabolize a range of brain neurotransmitters, whose dysregulation is closely associated with the development of various neurological disorders. This is why MAOs have been the central target in pharmacological interventions for neurodegeneration for more than 60 years. Still, existing drugs only address symptoms and not the cause of the disease, which underlines the need to develop more efficient inhibitors without adverse effects. Methods: Our drug design strategy relied on docking 25 organic scaffolds to MAO-B, which were extracted from the ChEMBL20 database with the highest cumulative counts of unique member compounds and bioactivity assays. The most promising candidates were substituted with the inactivating propargylamine group, while further affinity adjustment was made by its N-methylation. A total of 46 propargylamines were submitted to the docking and molecular dynamics simulations, while the best binders underwent mechanistic DFT analysis that confirmed the hydride abstraction mechanism of the covalent inhibition reaction. Results: We identified indole-2-propargylamine 4fH and indole-2-N-methylpropargylamine 4fMe as superior MAO-B binders over the clinical drugs rasagiline and selegiline. DFT calculations highlighted 4fMe as more potent over selegiline, evident in a reduced kinetic requirement (ΔΔG‡ = −2.5 kcal mol−1) and an improved reaction exergonicity (ΔΔGR = −4.3 kcal mol−1), together with its higher binding affinity, consistently determined by docking (ΔΔGBIND = −0.1 kcal mol−1) and MM-PBSA analysis (ΔΔGBIND = −1.5 kcal mol−1). Conclusions: Our findings strongly advocate 4fMe as an excellent drug candidate, whose synthesis and biological evaluation are highly recommended. Also, our results reveal the structural determinants that influenced the affinity and inhibition rates that should cooperate when designing further MAO inhibitors, which are of utmost significance and urgency with the increasing prevalence of brain diseases. [ABSTRACT FROM AUTHOR]

Recently, a single-neuron degeneration model has been proposed to understand the development of idiopathic Parkinson's disease based on (i) the extremely slow development of the degenerative process before the onset of motor symptoms and during the progression of the disease and (ii) the fact that it is triggered by an endogenous neurotoxin that does not have an expansive character, limiting its neurotoxic effect to single neuromelanin-containing dopaminergic neurons. It has been proposed that aminochrome is the endogenous neurotoxin that triggers the neurodegenerative process in idiopathic Parkinson's disease by triggering mitochondrial dysfunction, oxidative stress, neuroinflammation, dysfunction of both lysosomal and proteasomal protein degradation, endoplasmic reticulum stress and formation of neurotoxic alpha-synuclein oligomers. Aminochrome is an endogenous neurotoxin that is rapidly reduced by flavoenzymes and/or forms adducts with proteins, which implies that it is impossible for it to have a propagative neurotoxic effect on neighboring neurons. Interestingly, the enzymes DT-diaphorase and glutathione transferase M2-2 prevent the neurotoxic effects of aminochrome. Natural compounds present in fruits, vegetables and other plant products have been shown to activate the KEAP1/Nrf2 signaling pathway by increasing the expression of antioxidant enzymes including DT-diaphorase and glutathione transferase. This review analyzes the possibility of searching for natural compounds that increase the expression of DT-diaphorase and glutathione transferase through activation of the KEAP1/Nrf2 signaling pathway. [ABSTRACT FROM AUTHOR]

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives. [ABSTRACT FROM AUTHOR]

Formate oxidase (FOx), which contains 8-formyl flavin adenine dinucleotide (FAD), exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline (GMC) oxidoreductase enzymes that contain only the standard FAD cofactor. The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity. However, the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood, which impedes further utilization of FOx. In this study, we employ molecular dynamics simulation, QM/MM umbrella sampling simulation, enzyme activity assay, site-directed mutagenesis, and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD. Our results reveal that a catalytic water molecule, rather than any catalytic amino acids, serves as a general base to deprotonate the C8 methyl group on FAD, thus facilitating the formation of a quinone-methide tautomer intermediate. An oxygen molecule subsequently oxidizes this intermediate, resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−. During the oxidation of FAD to 8-formyl FAD, the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol, which corresponds to the required 14-hour transformation time observed experimentally. Further, the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues, R87 and S94, respectively. Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75% of the wild-type, while the mutation to histidine results in a complete loss of activity. Similarly, the mutant S94I also leads to the deactivation of enzyme. This dependency arises because the nucleophilic OH− group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94. These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD, thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties. [ABSTRACT FROM AUTHOR]

Covalent adducts of flavin cofactors with nucleophiles play an important role in non‐canonical function of flavoenzymes as well as in flavin‐based catalysis. Herein, the interaction of flavin derivatives including substituted flavins (isoalloxazines), 1,10‐ethylene‐bridged flavinium salts, and non‐substituted alloxazine and deazaflavin with selected nucleophiles was investigated using an experimental and computational approach. Triphenylphosphine or trimethylphosphine, 1‐nitroethan‐1‐ide, and methoxide were selected as representatives of neutral soft, anionic soft, and hard nucleophiles, respectively. The interactions were investigated using UV/Vis and 1H NMR spectroscopy as well as by DFT calculations. The position of nucleophilic attack estimated using the calculated Gibbs free energy values was found to correspond with the experimental data, favouring the addition of phosphine and 1‐nitroethan‐1‐ide into position N(5) and methoxide into position C(10a) of 1,10‐ethylene‐bridged flavinium salts. The calculated Gibbs free energy values were found to correlate with the experimental redox potentials of the flavin derivatives tested. These findings can be utilized as valuable tools for the design of artificial flavin‐based catalytic systems or investigating the mechanism of flavoenzymes. [ABSTRACT FROM AUTHOR]

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition. Here, the authors present the structure of inactive methylenetetrahydrofolate reductase, revealing linker-mediated allostery coupled with dual SAM binding and revealing distinct domain rearrangements. [ABSTRACT FROM AUTHOR]

The carboxyl‐terminal (C‐terminal) S‐[(Z)‐2‐aminovinyl]‐cysteine (AviCys) analogs have been identified in four families of ribosomally synthesized and post‐translationally modified peptides (RiPPs): lanthipeptides, linaridins, thioamitides, and lipolanthines. Within identified biosynthetic pathways, a highly reactive enethiol intermediate, formed through an oxidative decarboxylation catalyzed by a LanD‐like flavoprotein, can undergo two types of cyclization: a Michael addition with a dehydroamino acid or a coupling reaction initiated by a radical species. The collaborative actions of LanD‐like proteins with diverse enzymes involved in dehydration, dethiolation or cyclization lead to the construction of structurally distinct peptide natural products with analogous C‐terminal macrocyclic moieties. This concept summarizes existing knowledge regarding biosynthetic pathways of AviCys analogs to emphasize the diversity of biosynthetic mechanisms that paves the way for future genome mining explorations into diverse peptide natural products. [ABSTRACT FROM AUTHOR]

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•ˉ) during substrate oxidation. The O2•ˉ reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme’s log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•ˉ can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function. Photosynthetic organisms, fungi, and animals contain distinct pathways for vitamin C biosynthesis, but the final biosynthetic step consistently involves an oxidation reaction catalysed by the aldonolactone oxidoreductases. Here, the authors investigate the origin and evolution of the diversified activities and substrate preferences featured by these enzymes using different methods and find evidence that they share a common ancestor. [ABSTRACT FROM AUTHOR]

The isoalloxazine heterocycle of flavin cofactors reacts with various nucleophiles to form covalent adducts with important functions in enzymes. Molecular flavin models allow for the characterization of such adducts and the study of their properties. A fascinating set of reactions occurs when flavins react with hydroxide base, which leads to imidazolonequinoxalines, ring‐contracted flavins, with so far unexplored activity. We report a systematic study of the photophysical properties of this new chromophore by absorption and emission spectroscopy as well as cyclic voltammetry. Excited, ring‐contracted flavins are significantly stronger hydrogen atom abstractors when compared to the parent flavins, which allowed the direct trifluoromethylthiolation of aliphatic methine positions (bond dissociation energy (BDE) of 400.8 kJ mol−1). In an orthogonal activity, their increased triplet energy (E(S0←T1)=244 kJ mol−1) made sensitized reactions possible which exceeded the power of standard flavins. Combining both properties, ring‐contracted flavin catalysts enabled the one‐pot, five‐step transformation of α‐tropolone into trans‐3,4‐disubstituted cyclopentanones. We envision this new class of flavin‐derived chromophores to open up new modes of reactivity that are currently impossible with unmodified flavins. [ABSTRACT FROM AUTHOR]

Flavoenzymes can mediate a large variety of oxidation reactions through the activation of oxygen. However, the O2 activation chemistry of flavin enzymes is not yet fully exploited. Normally, the O2 activation occurs at the C4a site of the flavin cofactor, yielding the flavin C4a‐(hydro)hydroperoxyl species in monooxygenases or oxidases. Using extensive MD simulations, QM/MM calculations and QM calculations, our studies reveal the formation of the common nucleophilic species, Flavin‐N5OOH, in two distinct flavoenzymes (RutA and EncM). Our studies show that Flavin‐N5OOH acts as a powerful nucleophile that promotes C−N cleavage of uracil in RutA, and a powerful base in the deprotonation of substrates in EncM. We reason that Flavin‐N5OOH can be a common reactive species in the superfamily of flavoenzymes, which accomplish generally selective general base catalysis and C−X (X=N, S, Cl, O) cleavage reactions that are otherwise challenging with solvated hydroxide ion base. These results expand our understanding of the chemistry and catalysis of flavoenzymes. [ABSTRACT FROM AUTHOR]

High-fat diets (HFDs) are associated with the development of metabolic disorders. However, the type of dietary fat may also be a critical risk factor. The study aimed to determine the effect of three months of feeding HFDs with different fatty acid composition on oxidative stress markers and the proteome of mouse kidneys. The study involved 24 Swiss-Webster mice, which were divided into four groups (n = 6) fed for three months a standard chow (STD), an HFD rich in saturated fatty acids (SFA), and HFDs rich in polyunsaturated fatty acids with linoleic acid to a-linolenic acid ratios of 14:1 (HR) or 5:1 (LR). Feeding the SFA and HR diets increased triglyceride levels compared to the STD and LR groups. The HR group also had higher concentrations of thiobarbituric acid reactive substances compared to the other groups. There was no effect of HFD on cholesterol concentration and activity of antioxidant enzymes. The SFA diet increased the expression of acyl-CoA thioesterase 2 and D-lactate dehydrogenase, while decreasing that of apolipoprotein E. All HFDs led to downregulation of ATP synthase F1 subunit beta, mitochondrial, while only the LR diet increased the expression of persulphide dioxygenase ETHE1 and electron transfer flavoprotein subunit A, suggesting an enhanced oxidation of fatty acids. Ingestion of the SFA and HR diets caused downregulation of molecular chaperones and upregulation of the inflammation and apoptosis regulator peptidylprolyl isomerase A. In conclusion, feeding SFA and HR diets induces oxidative stress and proteome changes in the mouse kidney. The SFA diet most significantly influenced the expression pattern of proteins related to energetic metabolism. [ABSTRACT FROM AUTHOR]

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine. [ABSTRACT FROM AUTHOR]

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCE Pseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate’s side chain. Structural analysis of the refined HspB crystal structure, combined with state-ofthe- art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells. [ABSTRACT FROM AUTHOR]

Enzymes are increasingly recognized as valuable (bio)catalysts that complement existing synthetic methods. However, the range of biotransformations used in the laboratory is limited. Here we give an overview on the biosynthesis‐inspired discovery of novel biocatalysts that address various synthetic challenges. Prominent examples from this dynamic field highlight remarkable enzymes for protecting‐group‐free amide formation and modification, control of pericyclic reactions, stereoselective hetero‐ and polycyclizations, atroposelective aryl couplings, site‐selective C−H activations, introduction of ring strain, and N−N bond formation. We also explore unusual functions of cytochrome P450 monooxygenases, radical SAM‐dependent enzymes, flavoproteins, and enzymes recruited from primary metabolism, which offer opportunities for synthetic biology, enzyme engineering, directed evolution, and catalyst design. [ABSTRACT FROM AUTHOR]

Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals. By integrating in silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify enzymes that are active on glycerol. Three thermostable alditol oxidases from Actinobacteria Bacterium, Streptomyces thermoviolaceus, and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent thermostabilities with melting temperatures higher than 75 °C. Structural elucidation of the alditol oxidase from Actinobacteria Bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen bonds, a histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols. Key points: • Rapid pipeline designed to identify putative oxidases • Biochemical and structural characterization of alditol oxidases • Glycerol oxidation to more valuable derivatives [ABSTRACT FROM AUTHOR]

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti‐AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane‐bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one‐hundred nanomolar range and optimal binding under reducing conditions. 3D‐modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD‐dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes. [ABSTRACT FROM AUTHOR]

Adenine nucleotide translocator (ANT) is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane. It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm. The isoform ANT1 predominately expresses in cardiac and skeletal muscles. Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases. We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-deficient mice. Hearts and quadriceps resected from age-matched wild type (WT) and ANT1-deficient mice were snap-frozen in liquid nitrogen. The Chance redox scanner was utilized to perform 3D optical redox imaging. Each sample underwent scanning across 3–5 sections. Global averaging analysis showed no significant differences in the redox indices (NADH, flavin adenine dinucleotide containing-flavoproteins Fp, and the redox ratio Fp/(NADH+Fp) between WT and ANT1-deficient groups. However, quadriceps had higher Fp than hearts in both groups (p = 0. 0 0 0 4 and 0.01, respectively). Furthermore, the quadriceps were also more oxidized (a higher redox ratio) than hearts in WT group (p = 0. 0 0 4). NADH levels were similar in all cases. Our data suggest that under non-stressful physical condition, the ANT1-deficient muscle cells were in the same mitochondrial state as WT ones and that the significant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-deficient ones. Redox imaging of muscles under physical stress can be conducted in future. [ABSTRACT FROM AUTHOR]

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the d-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases. [ABSTRACT FROM AUTHOR]

Only a few studies have explored relationships between riboflavin intake and function and a few studies have examined the effects of supplements on various clinical or biochemical outcomes. None of these studies, however, make a useful contribution to understanding requirements in healthy populations. Thus, there is no strong evidence to change the recommendations.The requirement for riboflavin is estimated based on the relationship between intake and biochemical indices of riboflavin status, including urinary excretion and enzyme activities.

Adenine nucleotide translocator (ANT) is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane. It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm. The isoform ANT1 predominately expresses in cardiac and skeletal muscles. Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases. We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-deficient mice. Hearts and quadriceps resected from age-matched wild type (WT) and ANT1-deficient mice were snap-frozen in liquid nitrogen. The Chance redox scanner was utilized to perform 3D optical redox imaging. Each sample underwent scanning across 3–5 sections. Global averaging analysis showed no significant differences in the redox indices (NADH, flavin adenine dinucleotide containing-flavoproteins Fp, and the redox ratio Fp/(NADH+Fp) between WT and ANT1-deficient groups. However, quadriceps had higher Fp than hearts in both groups ([Formula: see text] and 0.01, respectively). Furthermore, the quadriceps were also more oxidized (a higher redox ratio) than hearts in WT group ([Formula: see text]). NADH levels were similar in all cases. Our data suggest that under non-stressful physical condition, the ANT1-deficient muscle cells were in the same mitochondrial state as WT ones and that the significant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-deficient ones. Redox imaging of muscles under physical stress can be conducted in future.

An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability of the two-electron reduced anionic FAD state (FADH−) in CraCRY and related (6–4) photolyases. The thermodynamic stability of FADH− remained almost the same compared to that of all tested proteins. However, the kinetic stability of FADH− varied remarkably depending on the local structure of the secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the kinetic stability of FADH− suggests an essential role of 8-HDF in the bifunctionality of CraCRY. [ABSTRACT FROM AUTHOR]

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients. Flavinated succinate dehydrogenase A (SDHA) is found to be responsible for the reduced autofluorescence observed in oral squamous cell carcinoma, and the functional metabolic implication of this observation is investigated. [ABSTRACT FROM AUTHOR]

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, H2O2 is likely to be the reactive agent to attack the substrate. The H2O2 assay results demonstrated production of H2O2 from the oxidation of FMNH−:DMSMO, whereas H2O2 was not detected in the presence of DMSO2, confirming H2O2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H2O2 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. [ABSTRACT FROM AUTHOR]

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5‐deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one‐electron‐transfer reactions in flavoproteins are impeded with 5‐deazaflavin as the cofactor. Based on these findings, it was concluded that the 5‐deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re‐oxidizes or undergoes disproportionation. The long‐standing paradigm of 5‐deazaflavin being solely a two‐electron/hydride acceptor/donor—"a nicotinamide in flavin clothing"—needs to be re‐evaluated now with the indirect observation of a one‐electron‐reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo‐CIDNP) 1H nuclear magnetic resonance (NMR) under biologically relevant conditions. [ABSTRACT FROM AUTHOR]

Electron transfer flavoprotein subunit beta (ETFB) of Leptospira interrogans is a biomarker for diagnosing leptospiral infection. Thus, the ETFB-specific nuclease-resistant RNA aptamer ETFB3-63 was developed and used in an electrochemical aptasensor to assay ETFB. Although the majority of reported biosensors detect various genes and antibodies of L. interrogans, this is the first attempt to construct an electrochemical biosensor to detect ETFB protein for the diagnosis of leptospiral infection. The ETFB protein can be detected without any extraction phase. In this assay, a single-stranded DNA probe complementary to the ETFB3-63 sequence was immobilized on a screen-printed carbon electrode (SPCE). The aptamer was then incubated and hybridized with the antisense probe on the SPCE. In the presence of ETFB, the aptamer dissociates from the aptamer/probe complex on the SPCE to bind with the protein. Methylene blue was then added to intercalate with the remaining hybridized aptamers, and its signal was measured using differential pulse voltammetry. The signal arising from the intercalated methylene blue decreased with increasing concentration of ETFB, showing a linear response in the range of 50–500 nM of ETFB and 10 to 109 leptospira cells per mL, respectively. The aptasensor signal was also specific to L. interrogans but not to 12 related bacteria tested. In addition, the aptasensor showed similar performance in detecting ETFB spiked in human serum to that in buffer, indicating that proteins in the serum do not interfere with the assay. Therefore, this assay has great potential to develop into a point-of-care electrochemical device that is accurate, cost-effective, and user-friendly for leptospirosis diagnosis. [ABSTRACT FROM AUTHOR]

FLAD1, along with its FAD synthase (FADS, EC 2.7.7.2) product, is crucial for flavin homeostasis and, due to its role in the mitochondrial respiratory chain and nuclear epigenetics, is closely related to cellular metabolism. Therefore, it is not surprising that it could be correlated with cancer. To our knowledge, no previous study has investigated FLAD1 prognostic significance in pancreatic ductal adenocarcinoma (PDAC). Thus, in the present work, the FAD synthesis process was evaluated in two PDAC cell lines: (a) PANC‐1‐ and PANC‐1‐derived cancer stem cells (CSCs), presenting the R273H mutation in the oncosuppressor p53, and (b) MiaPaca2 and MiaPaca2‐derived CSCs, presenting the R248W mutation in p53. As a control, HPDE cells expressing wt‐p53 were used. FADS expression/activity increase was found with malignancy and even more with stemness. An increased FAD synthesis rate in cancer cell lines is presumably demanded by the increase in the FAD‐dependent lysine demethylase 1 protein amount as well as by the increased expression levels of the flavoprotein subunit of complex II of the mitochondrial respiratory chain, namely succinate dehydrogenase. With the aim of proposing FADS as a novel target for cancer therapy, the inhibitory effect of Chicago Sky Blue on FADS enzymatic activity was tested on the recombinant 6His‐hFADS2 (IC50 = 1.2 μm) and PANC‐1‐derived CSCs' lysate (IC50 = 2–10 μm). This molecule was found effective in inhibiting the growth of PANC‐1 and even more of its derived CSC line, thus assessing its role as a potential chemotherapeutic drug. [ABSTRACT FROM AUTHOR]

The mechanisms initiating apoptotic programmed cell death in diverse eukaryotes are very similar. Basically, the mitochondrial permeability transition activates apoptotic proteases, DNases, and flavoproteins such as apoptosis-inducing factors (AIFs). According to the hypothesis of the endosymbiotic origin of apoptosis, these mechanisms evolved during mitochondrial domestication. Various phylogenetic analyses, including ours, have suggested that apoptotic factors were eubacterial protomitochondrial toxins used for killing protoeukaryotic hosts. Here, we tested whether the function of yeast Saccharomyces cerevisiae apoptotic proteases (metacaspases Mca1 and Nma111), DNase Nuc1, and flavoprotein Ndi1 can be substituted with orthologs from remotely related eukaryotes such as plants, protists, and eubacteria. We found that orthologs of remotely related eukaryotic and even eubacterial proteins can initiate apoptosis in yeast when triggered by chemical stresses. This observation suggests that apoptotic mechanisms have been maintained since mitochondrial domestication, which occurred approximately 1,800 Mya. Additionally, it supports the hypothesis that some of these apoptotic factors could be modified eubacterial toxins. [ABSTRACT FROM AUTHOR]

Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.

Cryptochromes are flavoproteins related to photolyases that are widespread throughout the plant and animal kingdom. They govern blue light-dependent growth in plants, control circadian rhythms in a light-dependent manner in invertebrates, and play a central part in the circadian clock in vertebrates. In addition, cryptochromes might function as receptors that allow animals to sense the Earth's magnetic field. As cryptochromes are also present in mammals including humans, the possibility of a magnetosensitive protein is exciting. Here we attempt to provide a concise overview of cryptochromes in mammals. We briefly review their canonical role in the circadian rhythm from the molecular level to physiology, behaviour and diseases. We then discuss their disputed light sensitivity and proposed role in the magnetic sense in mammals, providing three mechanistic hypotheses. Specifically, mammalian cryptochromes could form light-induced radical pairs in particular cellular milieus, act as magnetoreceptors in darkness, or as secondary players in a magnetoreception signalling cascade. Future research can test these hypotheses to investigate if the role of mammalian cryptochromes extends beyond the circadian clock. [ABSTRACT FROM AUTHOR]

Photolyases are flavoproteins, which are able to repair UV‐induced DNA lesions in a light‐dependent manner. According to their substrate, they can be distinguished as CPD‐ and (6‐4) photolyases. While CPD‐photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6‐4) photolyases catalyze the repair of the less prominent (6‐4) photoproduct. The subgroup of prokaryotic (6‐4) photolyases/FeS‐BCP is one of the most ancient types of flavoproteins in the ubiquitously occurring photolyase & cryptochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6‐4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential flavin adenine dinucleotide as well as an elongated linker region between the N‐terminal α/β‐domain and the C‐terminal all‐α‐helical domain. Furthermore, they can harbor an additional short subdomain, located at the C‐terminus, with a binding site for a [4Fe‐4S] cluster. So far, two crystal structures of prokaryotic (6‐4) photolyases have been reported. Within this study, we present the high‐resolution structure of the prokaryotic (6‐4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA‐repair activity. [ABSTRACT FROM AUTHOR]

The benefits of exercise involve skeletal muscle redox state alterations of nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). We determined the fiber-specific effects of acute exercise on the skeletal muscle redox state in healthy adults. Muscle biopsies were obtained from 19 participants (11 M, 8 F; 26 ± 4 yr) at baseline (fasted) and 30 min and 3 h after treadmill exercise at 80% maximal oxygen consumption (VO2max). Muscle samples were probed for autofluorescence of NADH (excitation at 340-360 nm) and oxidized flavoproteins (Fp; excitation at 440-470 nm) and subsequently, fiber typed to quantify the redox signatures of individual muscle fibers. Redox state was calculated as the oxidation-to-reduction redox ratio: Fp/(Fp + NADH). At baseline, pair-wise comparisons revealed that the redox ratio of myosin heavy chain (MHC) I fibers was 7.2% higher than MHC IIa (P = 0.023, 95% CI: 5.2, 9.2%) and the redox ratio of MHC IIa was 8.0% higher than MHC IIx (P = 0.035, 95% CI: 6.8, 9.2%). MHC I fibers also displayed greater NADH intensity than MHC IIx (P = 0.007) and greater Fp intensity than both MHC IIa (P = 0.019) and MHC IIx (P < 0.0001). Fp intensities increased in all fiber types (main effect, P = 0.039) but redox ratios did not change (main effect, P ¼ 0.483) 30 min after exercise. The change in redox ratio was positively correlated with capillary density in MHC I (rho = 0.762, P = 0.037), MHC IIa fibers (rho = 0.881, P = 0.007), and modestly in MHC IIx fibers (rho = 0. 771, P ¼ 0.103). These findings support the use of redox autofluorescence to interrogate skeletal muscle metabolism. [ABSTRACT FROM AUTHOR]

Photolyases are flavoproteins, which are able to repair UV‐induced DNA lesions in a light‐dependent manner. According to their substrate, they can be distinguished as CPD‐ and (6‐4) photolyases. While CPD‐photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6‐4) photolyases catalyze the repair of the less prominent (6‐4) photoproduct. The subgroup of prokaryotic (6‐4) photolyases/FeS‐BCP is one of the most ancient types of flavoproteins in the ubiquitously occurring photolyase & cryptochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6‐4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential flavin adenine dinucleotide as well as an elongated linker region between the N‐terminal α/β‐domain and the C‐terminal all‐α‐helical domain. Furthermore, they can harbor an additional short subdomain, located at the C‐terminus, with a binding site for a [4Fe‐4S] cluster. So far, two crystal structures of prokaryotic (6‐4) photolyases have been reported. Within this study, we present the high‐resolution structure of the prokaryotic (6‐4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA‐repair activity. [ABSTRACT FROM AUTHOR]

LanD flavoproteins catalyze oxidative decarboxylation of the C‐terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S‐[2‐aminovinyl](3‐methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C‐terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two‐stage bioinformatics mining of posttranslational modifications (PTMs) related to C‐terminal Cys processing, we report herein that LanD activity can couple with radical S‐adenosylmethionine chemistry to provide a new unsaturated thioether residue, S‐[2‐aminovinyl]‐3‐carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C‐terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs. [ABSTRACT FROM AUTHOR]

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes. [ABSTRACT FROM AUTHOR]

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARa, including those required for gluconeogenesis. We also found PPARa knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARa agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs. [ABSTRACT FROM AUTHOR]

The flavin‐dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7‐dibromotryptophan. In contrast to the well studied two‐component tryptophan halogenases, AetF is a single‐component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single‐component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin‐dependent monooxygenases. It contains two dinucleotide‐binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5‐bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5‐bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7‐bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single‐component FDHs. [ABSTRACT FROM AUTHOR]

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin‐dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions—but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C‐terminal 23 residues (the 'α‐peptide'). Here, we relate the redox‐induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (−SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys–SH difference signal to Cys88 and Cys494, both being remote from the moving α‐peptide and the redox‐active flavin cofactor. Yet, when the α‐peptide is proteolytically removed, the Cys–SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α‐peptide 'transforms' the catalytically complex EcPOX into the catalytically 'simpler' LpPOX. [ABSTRACT FROM AUTHOR]

The blue-light sensors, cryptochromes, compose the extensive class of flavoprotein photoreceptors, regulating signaling processes in plants underlying their development, growth, and metabolism. In several algae, cryptochromes may act not only as sensory photoreceptors but also as photolyases, catalyzing repair of the UV-induced DNA lesions. Cryptochromes bind FAD as the chromophore at the photolyase homologous region (PHR) domain and contain the cryptochrome C-terminal extension (CCE), which is absent in photolyases. Photosensory process in cryptochrome is initiated by photochemical chromophore conversions, including formation of the FAD redox forms. In the state with the chromophore reduced to neutral radical (FADH•), the photoreceptor protein undergoes phosphorylation, conformational changes, and disengagement from the PHR domain and CCE with subsequent formation of oligomers of cryptochrome molecules. Photooligomerization is a structural basis of the functional activities of cryptochromes, since it ensures formation of their complexes with a variety of signaling proteins, including transcriptional factors and regulators of transcription. Interactions in such complexes change the protein signaling activities, leading to regulation of gene expression and plant photomorphogenesis. In recent years, multiple papers, reporting novel, more detailed information about the molecular mechanisms of above-mentioned processes were published. The present review mainly focuses on analysis of the data contained in these publications, particularly regarding structural aspects of the cryptochrome transitions into photoactivated states and regulatory signaling processes mediated by the cryptochrome photoreceptors in plants. [ABSTRACT FROM AUTHOR]

Background: Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. Results: In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. Conclusion: The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Numerous bacteria from different phylae can perform desulfurization reactions of organosulfur compounds. In these degradation or detoxification pathways, two‐component flavin‐dependent monooxygenases that use flavins (FMN or FAD) as a cofactor play important roles as they catalyse the first steps of these metabolic routes. The TdsC or DszC and MsuC proteins belong to this class of enzymes as they process dibenzothiophene (DBT) and methanesulfinate. Elucidation of their X‐ray structures in apo, ligand‐bound and cofactor‐bound forms has provided important molecular insights into their catalytic reaction. Mycobacterial species have also been shown to possess a DBT degradation pathway, but no structural information is available on these two‐component flavin‐dependent monooxygenases. In this study, the crystal structure of the uncharacterized MAB_4123 protein from the human pathogen Mycobacterium abscessus is presented. The structure solved at high resolution displays high similarity to homologs from Rhodococcus, Paenibacillus and Pseudomonas species. In silico docking approaches suggest that MAB_4123 binds FMN and may use it as a cofactor. Structural analysis strongly suggests that MAB_4123 is a two‐component flavin‐dependent monooxygenase that could act as a detoxifying enzyme of organosulfur compounds in mycobacteria. [ABSTRACT FROM AUTHOR]

A multifunctional hybrid constructed for controlling the delivery and activation of Pt anticancer agents in vitro is described herein. We employed consensus tetratricopeptide repeat protein (CTPR) for the covalent co-anchoring of riboflavin (photocatalyst) and a Pt(IV) prodrug complex. The Pt-loaded flavoprotein induced a 40% reduction in PANC-1 cell viability as a result of the photocatalytic formation of cisplatin. [ABSTRACT FROM AUTHOR]

Flavoprotein‐Monooxygenasen sind eine vielseitige Gruppe von Enzymen für biokatalytische Reaktionen. Die dazugehörigen Monooxygenasen der Gruppe E (GEMs) katalysieren enantioselektive Epoxidierungs‐ und Sulfoxidierungsreaktionen. In dieser Arbeit beschreiben wir die Kristallstruktur einer Indol‐Monooxygenase aus dem Bakterium Variovorax paradoxus EPS, einer GEM mit der Bezeichnung VpIndA1. Basierend auf Substratkomplexstrukturen produktiver Bindungsmodi und in Verbindung mit Kraftfeldberechnungen sowie durch schnelle Mischungskinetik zeigen wir die strukturelle Grundlage der Substrat‐ und Stereoselektivität auf. Die strukturbasierte Umgestaltung der Substrattasche führte zu Varianten mit neuer Substratselektivität (für die Sulfoxidation von Benzylphenylsulfid) oder mit stark erhöhter Stereoselektivität (von 35.1 % auf 99.8 % ee für die Herstellung von (1S,2R)‐Indenoxid). Diese erste Bestimmung des Substratbindungsmodus von GEMs in Verbindung mit der Untersuchung von Struktur‐Funktions‐Beziehungen öffnet die Tür für ein strukturbasiertes Design dieser leistungsstarken Biokatalysatoren. [ABSTRACT FROM AUTHOR]

Photoreceptors containing the light‐oxygen‐voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark‐state recovery are not well understood. Here we incorporated the non‐canonical amino acid p‐cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light‐induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time‐resolved multi‐probe UV/visible and IR spectroscopy experiments of the lit‐to‐dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN‐cysteinyl adduct scission, folding of α‐helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N‐terminal A'α extension of the LOV domain in controlling EL222 photocycle length. [ABSTRACT FROM AUTHOR]