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1.
J Chem Inf Model ; 64(12): 4773-4780, 2024 Jun 24.
Article En | MEDLINE | ID: mdl-38837697

Lysine-specific demethylase 1 (LSD1), a highly sophisticated epigenetic regulator, orchestrates a range of critical cellular processes, holding promising therapeutic potential for treating diverse diseases. However, the clinical research progress targeting LSD1 is very slow. After 20 years of research, only one small-molecule drug, BEA-17, targeting the degradation of LSD1 and CoREST has been approved by the U.S. Food and Drug Administration. The primary reason for this may be the lack of abundant structural data regarding its intricate functions. To gain a deeper understanding of its conformational dynamics and guide the drug design process, we conducted molecular dynamics simulations to explore the conformational states of LSD1 in the apo state and under the influence of cofactors of flavin adenine dinucleotide (FAD) and CoREST. Our results showed that, across all states, the substrate binding pocket exhibited high flexibility, whereas the FAD binding pocket remained more stable. These distinct dynamical properties are essential for LSD1's ability to bind various substrates while maintaining efficient demethylation activity. Both pockets can be enlarged by merging with adjacent pockets, although only the substrate binding pocket can shrink into smaller pockets. These new pocket shapes can inform inhibitor design, particularly for selectively FAD-competitive inhibitors of LSD1, given the presence of numerous FAD-dependent enzymes in the human body. More interestingly, in the absence of FAD binding, the united substrate and FAD binding pocket are partitioned by the conserved residue of Tyr761, offering valuable insights for the design of inhibitors that disrupt the crucial steric role of Tyr761 and the redox role of FAD. Additionally, we identified pockets that positively or negatively correlate with the substrate and FAD binding pockets, which can be exploited for the design of allosteric or concurrent inhibitors. Our results reveal the intricate dynamical properties of LSD1 as well as multiple novel conformational states, which deepen our understanding of its sophisticated functions and aid in the rational design of new inhibitors.


Drug Design , Enzyme Inhibitors , Flavin-Adenine Dinucleotide , Histone Demethylases , Molecular Dynamics Simulation , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/metabolism , Histone Demethylases/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Binding Sites , Humans , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Substrate Specificity , Protein Conformation , Protein Binding
2.
Nat Commun ; 15(1): 5167, 2024 Jun 17.
Article En | MEDLINE | ID: mdl-38886362

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.


Chaetomium , Methylenetetrahydrofolate Reductase (NADPH2) , S-Adenosylmethionine , Methylenetetrahydrofolate Reductase (NADPH2)/metabolism , Methylenetetrahydrofolate Reductase (NADPH2)/chemistry , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , S-Adenosylmethionine/metabolism , S-Adenosylmethionine/chemistry , Allosteric Regulation , Chaetomium/enzymology , Chaetomium/metabolism , Chaetomium/genetics , Phosphorylation , Humans , Crystallography, X-Ray , Models, Molecular , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry
3.
J Phys Chem B ; 128(22): 5320-5326, 2024 Jun 06.
Article En | MEDLINE | ID: mdl-38805723

It is still a puzzle that has not been entirely solved how migratory birds utilize the Earth's magnetic field for biannual migration. The most consistent explanation thus far is rooted in the modulation of the biological function of the cryptochrome 4 (Cry4) protein by an external magnetic field. This phenomenon is closely linked with the flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the protein. Cry4 is activated by blue light, which is absorbed by the FAD cofactor. Subsequent electron and proton transfers trigger radical pair formation in the protein, which is sensitive to the external magnetic field. An important long-lasting redox state of the FAD cofactor is the signaling (FADH•) state, which is present after the transient electron transfer steps have been completed. Recent experimental efforts succeeded in crystallizing the Cry4 protein from Columbia livia (ClCry4) with all of the important residues needed for protein photoreduction. This specific crystallization of Cry4 protein so far is the only avian cryptochrome crystal structure available, which, however, has great similarity to the Cry4 proteins of night migratory birds. The previous experimental studies of the ClCry4 protein included the absorption properties of the protein in its different redox states. The absorption spectrum of the FADH• state demonstrated a peculiar red shift compared to the photoabsorption properties of the FAD cofactor in its FADH• state in other Cry proteins from other species. The aim of this study is to understand this red shift by employing the tools of computational microscopy and, in particular, a QM/MM approach that relies on the polarizable embedding approximation.


Cryptochromes , Flavin-Adenine Dinucleotide , Cryptochromes/chemistry , Cryptochromes/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Animals , Avian Proteins/chemistry , Avian Proteins/metabolism , Oxidation-Reduction
4.
Nat Commun ; 15(1): 3994, 2024 May 11.
Article En | MEDLINE | ID: mdl-38734761

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.


Calcium , Cryoelectron Microscopy , Molecular Dynamics Simulation , NADPH Oxidase 5 , NADP , Humans , NADPH Oxidase 5/metabolism , NADPH Oxidase 5/genetics , NADPH Oxidase 5/chemistry , Calcium/metabolism , NADP/metabolism , Flavin-Adenine Dinucleotide/metabolism , Superoxides/metabolism , Protein Binding , Reactive Oxygen Species/metabolism , Zinc/metabolism , Electron Transport , Enzyme Activation , Binding Sites
5.
Biochemistry ; 63(10): 1347-1358, 2024 May 21.
Article En | MEDLINE | ID: mdl-38691339

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.


Dihydroorotate Dehydrogenase , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Lactococcus lactis/enzymology , Lactococcus lactis/metabolism , Oxidation-Reduction , Catalytic Domain , Kinetics , Flavin Mononucleotide/metabolism , Flavin Mononucleotide/chemistry , NAD/metabolism , NAD/chemistry , Catalysis , Flavins/metabolism , Biocatalysis , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry
6.
Int J Mol Sci ; 25(10)2024 May 14.
Article En | MEDLINE | ID: mdl-38791410

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be -0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.


Bacillus subtilis , Ferredoxin-NADP Reductase , Oxidation-Reduction , Ferredoxin-NADP Reductase/metabolism , Ferredoxin-NADP Reductase/chemistry , Bacillus subtilis/enzymology , Xenobiotics/metabolism , Xenobiotics/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Potentiometry , Oxidants/chemistry , Quinones/metabolism , Quinones/chemistry , Electron Transport
7.
Phys Chem Chem Phys ; 26(23): 16521-16528, 2024 Jun 12.
Article En | MEDLINE | ID: mdl-38809594

Indole monooxygenases (IMOs) are enzymes from the family of Group E monooxygenases, requiring flavin adenine dinucleotide (FAD) for their activities. IMOs play important roles in both sulfoxidation and epoxidation reactions. The broad substrate range and high selectivity of IMOs make them promising biocatalytic tools for synthesizing chiral compounds. In the present study, quantum chemical calculations using the cluster approach were performed to investigate the reaction mechanism and the enantioselectivity of the IMO from Variovorax paradoxus EPS (VpIndA1). The sulfoxidation of methyl phenyl sulfide (MPS) and the epoxidation of indene were chosen as the representative reactions. The calculations confirmed that the FADOOH intermediate is the catalytic species in the VpIndA1 reactions. The oxidation of MPS adopts a one-step mechanism involving the direct oxygen-transfer from FADOOH to the substrate and the proton transfer from the -OH group back to FAD, while the oxidation of indene follows a stepwise mechanism involving a carbocation intermediate. It was computationally predicted that VpIndA1 prefers the formation of (S)-product for the MPS sulfoxidation and (1S,2R)-product for the indene epoxidation, consistent with the experimental observations. Importantly, the factors controlling the stereo-preference of the two reactions are identified. The findings in the present study provide valuable insights into the VpIndA1-catalyzed reactions, which are essential for the rational design of this enzyme and other IMOs for industrial applications. It is also worth emphasizing that the quantum chemical cluster approach is again demonstrated to be powerful in studying the enantioselectivity of enzymatic reactions.


Mixed Function Oxygenases , Oxidation-Reduction , Stereoisomerism , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/chemistry , Quantum Theory , Sulfides/chemistry , Sulfides/metabolism , Indoles/chemistry , Indoles/metabolism , Models, Chemical , Epoxy Compounds/chemistry , Epoxy Compounds/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Models, Molecular
8.
Biochemistry (Mosc) ; 89(2): 241-256, 2024 Feb.
Article En | MEDLINE | ID: mdl-38622093

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.


Oxidoreductases , Vibrio , Oxidoreductases/metabolism , NAD/metabolism , Cinnamates , Oxidation-Reduction , Vibrio/genetics , Vibrio/metabolism , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , NADH Dehydrogenase/metabolism , Flavins/chemistry , Transferases , Flavin-Adenine Dinucleotide/metabolism
9.
Comput Biol Chem ; 110: 108075, 2024 Jun.
Article En | MEDLINE | ID: mdl-38678729

Glutathione reductase (GR) is a two dinucleotide binding domain flavoprotein (tDBDF) that catalyzes the reduction of glutathione disulfide to glutathione coupled to the oxidation of NADPH to NADP+. An interesting feature of GR and other tDBDFs is the presence of a lysine residue (Lys-66 in human GR) at the active site, which interacts with the flavin group, but has an unknown function. To better understand the role of this residue, the dynamics of GR was studied using molecular dynamics simulations, and the reaction mechanism of FAD reduction by NADPH was studied using QM/MM molecular modeling. The two possible protonation states of Lys-66 were considered: neutral and protonated. Molecular dynamics results suggest that the active site is more structured for neutral Lys-66 than for protonated Lys-66. QM/MM modeling results suggest that Lys-66 should be in its neutral state for a thermodynamically favorable reduction of FAD by NADPH. Since the reaction is unfavorable with protonated Lys-66, the reverse reaction (the reduction of NADP+ by FADH-) is expected to take place. A phylogenetic analysis of various tDBDFs was performed, finding that an active site lysine is present in different the tDBDFs enzymes, suggesting that it has a conserved biological role. Overall, these results suggest that the protonation state of the active site lysine determines the energetics of the reaction, controlling its reversibility.


Catalytic Domain , Flavin-Adenine Dinucleotide , Glutathione Reductase , Lysine , Molecular Dynamics Simulation , NADP , Oxidation-Reduction , Lysine/chemistry , Lysine/metabolism , NADP/metabolism , NADP/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Humans , Glutathione Reductase/metabolism , Glutathione Reductase/chemistry , Quantum Theory
10.
Cell Rep ; 43(5): 114130, 2024 May 28.
Article En | MEDLINE | ID: mdl-38640062

Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically active during their evolution is still an open question. Two opposite explanations are plausible: acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event. Here, we use molecular phylogeny, ancestral sequence reconstruction, and biochemical characterization to follow the evolution of a specialized group of flavoprotein monooxygenases, the bacterial Baeyer-Villiger monooxygenases (BVMOs). These enzymes catalyze an intricate chemical reaction relying on three different elements: a reduced nicotinamide cofactor, dioxygen, and a substrate. Characterization of ancestral BVMOs shows that the catalytic mechanism evolved in a series of steps starting from a FAD-binding protein and further acquiring reactivity and specificity toward each of the elements participating in the reaction. Together, the results of our work portray how an intrinsically complex catalytic mechanism emerged during evolution.


Evolution, Molecular , Mixed Function Oxygenases , Phylogeny , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/chemistry , Catalysis , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Biocatalysis , Flavin-Adenine Dinucleotide/metabolism , Substrate Specificity , Oxygen/metabolism
11.
J Neurochem ; 168(5): 663-676, 2024 May.
Article En | MEDLINE | ID: mdl-38439211

Neurons exhibit a high energetic need, and the question arises as how they metabolically adapt to changing activity states. This is relevant for interpreting functional neuroimaging in different brain areas. Particularly, neurons with a broad firing range might exhibit metabolic adaptations. Therefore, we studied MNTB (medial nucleus of the trapezoid body) principal neurons, which generate action potentials (APs) at frequencies up to several hundred hertz. We performed the experiments in acute brainstem slices of the Mongolian gerbil (Meriones unguiculatus) at 22.5-24.5°C. Upon electrical stimulation of afferent MNTB fibres with 400 stimuli at varying frequencies, we monitored autofluorescence levels of NAD(P)H and FAD and determined the extremum amplitudes of their biphasic response. Additionally, we compared these data with alterations in O2 concentrations measured with an electrochemical sensor. These O2 changes are prominent since MNTB neurons rely on oxidative phosphorylation as shown by our pharmacological experiments. We calculated the O2 consumption rate as change in O2 concentration divided by stimulus durations, because these periods varied inversely with stimulus frequency as a result of the constant number of 400 stimuli applied. The O2 consumption rate increased with stimulation frequency up to a constant value at 600 Hz; that is, energy demand depends on temporal characteristics of activity despite the same number of stimuli. The rates showed no correlation with peak amplitudes of NAD(P)H or FAD, whilst the values of the two molecules were linearly correlated. This points at the complexity of analysing autofluorescence imaging for quantitative metabolic studies, because these values report only relative net changes of many superimposed oxidative and reductive processes. Monitoring O2 concentration rates is, thus, an important tool to improve the interpretation of NAD(P)H/FAD autofluorescence data, as they do not under all conditions and in all systems appropriately reflect the metabolic activity or energy demand.


Brain Stem , Gerbillinae , Neurons , Animals , Neurons/metabolism , Neurons/physiology , Brain Stem/metabolism , Oxygen Consumption/physiology , Action Potentials/physiology , Male , Electric Stimulation , Flavin-Adenine Dinucleotide/metabolism , Female , Trapezoid Body/physiology , Trapezoid Body/metabolism , NADP/metabolism
12.
J Biol Chem ; 300(5): 107238, 2024 May.
Article En | MEDLINE | ID: mdl-38552736

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.


Alternaria , Blue Light , Flavin-Adenine Dinucleotide , Fungal Proteins , Photoreceptors, Microbial , Alternaria/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Fungal Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/chemistry , Gene Expression Regulation, Fungal , Photoreceptors, Microbial/metabolism , Photoreceptors, Microbial/chemistry , Photoreceptors, Microbial/genetics , Phytochrome/metabolism , Phytochrome/chemistry , Phytochrome/genetics , Protein Domains , Reactive Oxygen Species/metabolism , Temperature
13.
Chembiochem ; 25(7): e202300833, 2024 Apr 02.
Article En | MEDLINE | ID: mdl-38306174

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO - a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45-50 Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (kred=110 s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox=90 s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec=0.2 s-1) compared to free cofactor (kdec=1.8 s-1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120 s-1. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO's ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH2 channelling between subunits. This work contributes to comprehending FADH2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.


Hydrogen Peroxide , Oxygenases , Oxygenases/metabolism , Styrene , Computer Simulation , Kinetics , Flavin-Adenine Dinucleotide/metabolism
14.
Appl Environ Microbiol ; 90(3): e0225523, 2024 Mar 20.
Article En | MEDLINE | ID: mdl-38415602

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.IMPORTANCEPseudomonas 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-of-the-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.


Mixed Function Oxygenases , Nicotine , Succinates , Mixed Function Oxygenases/metabolism , Nicotine/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavoproteins/metabolism , Hydroxylation , Pyridines/metabolism
15.
Nature ; 627(8002): 189-195, 2024 Mar.
Article En | MEDLINE | ID: mdl-38355798

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.


NADPH Oxidase 2 , Phagocytes , Humans , Electrons , Enzyme Activation , Flavin-Adenine Dinucleotide/metabolism , Heme/chemistry , Heme/metabolism , NADP/metabolism , NADPH Oxidase 2/chemistry , NADPH Oxidase 2/metabolism , Phagocytes/enzymology , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Superoxides/metabolism , Protein Binding
16.
ACS Appl Mater Interfaces ; 16(5): 5504-5512, 2024 Feb 07.
Article En | MEDLINE | ID: mdl-38278768

New insights into the unique biochemical properties of riboflavin (Rf), also known as vitamin B2, are leading to the development of its use not only as a vitamin supplement but also as a potential anti-inflammatory, immunomodulatory, antioxidant, anticancer, and antiviral agent, where it may play a role as an inhibitor of viral proteinases. At the same time, the comparison of the pharmacoactivity of Rf with its known metabolites, namely, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is very complicated due to its poor water solubility: 0.1-0.3 g/L versus 67 g/L for FMN and 50 g/L for FAD, which is the limiting factor for its administration in clinical practice. In this study, we report the recrystallization procedure of the type A Rf crystals into the slightly hydrophobic type B/C and a new hydrophilic crystal form that has been termed the P type. Our method of Rf crystal modification based on recrystallization from dilute alkaline solution provides an unprecedented extremely high water solubility of Rf, reaching 23.5 g/L. A comprehensive study of the physicochemical properties of type P riboflavin showed increased photodynamic therapeutic activity compared to the known types A and B/C against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium. Importantly, our work not only demonstrates a simple and inexpensive method for the synthesis of riboflavin with high solubility, which should lead to increased bioactivity, but also opens up opportunities for improving both known and new therapeutic applications of vitamin B2.


Flavin Mononucleotide , Flavin-Adenine Dinucleotide , Flavin-Adenine Dinucleotide/metabolism , Solubility , Riboflavin , Escherichia coli/metabolism , Water
17.
FEBS J ; 291(1): 177-203, 2024 01.
Article En | MEDLINE | ID: mdl-37786987

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.


Escherichia coli Proteins , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavoproteins/metabolism , Oxidoreductases/metabolism , Ligands , Escherichia coli/genetics , Escherichia coli/metabolism , Brain/metabolism , Endothelium/metabolism , Bacterial Adhesion
18.
Environ Res ; 242: 117712, 2024 Feb 01.
Article En | MEDLINE | ID: mdl-37993045

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.


Dinitrocresols , Flavin-Adenine Dinucleotide , Sewage , Flavin-Adenine Dinucleotide/metabolism , Flavin Mononucleotide/metabolism , Electrons , Anaerobiosis , Extracellular Polymeric Substance Matrix/metabolism , Riboflavin/metabolism , Dietary Supplements , Methane
19.
J Biol Chem ; 300(1): 105470, 2024 Jan.
Article En | MEDLINE | ID: mdl-38118236

The prevailing notion that reduced cofactors NADH and FADH2 transfer electrons from the tricarboxylic acid cycle to the mitochondrial electron transfer system creates ambiguities regarding respiratory Complex II (CII). CII is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the electron transfer system of the mitochondrial inner membrane feeding electrons into the coenzyme Q-junction. The succinate dehydrogenase subunit SDHA of CII oxidizes succinate and reduces the covalently bound prosthetic group FAD to FADH2 in the canonical forward tricarboxylic acid cycle. However, several graphical representations of the electron transfer system depict FADH2 in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH2 from the ß-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature and educational resources call for quality control, to secure scientific standards in current communications of bioenergetics, and ultimately support adequate clinical applications. This review aims to raise awareness of the inherent ambiguity crisis, complementing efforts to address the well-acknowledged issues of credibility and reproducibility.


Electron Transport Complex II , Electrons , Fatty Acids , Flavin-Adenine Dinucleotide , Succinate Dehydrogenase , Electron Transport , Fatty Acids/chemistry , Fatty Acids/metabolism , Flavin-Adenine Dinucleotide/analogs & derivatives , Flavin-Adenine Dinucleotide/metabolism , Oxidation-Reduction , Reproducibility of Results , Succinate Dehydrogenase/metabolism , Citric Acid Cycle , Mitochondria/metabolism , Ubiquinone/metabolism , Succinic Acid/metabolism , Electron Transport Complex II/metabolism , Energy Metabolism
20.
Biochim Biophys Acta Gen Subj ; 1868(1): 130520, 2024 01.
Article En | MEDLINE | ID: mdl-37952565

Flavin adenine dinucleotide (FAD) autofluorescence from cells reports on the enzymatic activity which involves FAD as a cofactor. Most of the cellular FAD fluorescence comes from complex II of the electron transport chain in mitochondria and can be assessed with inhibitor analysis. The intensity of FAD autofluorescence is not homogeneous and vary between cells in tissue and in cell culture types. Using primary co-culture of neurons and astrocytes, and human skin fibroblasts we have found that very high FAD autofluorescence is a result of an overactivation of the mitochondrial complex II from ETC and from the activity of monoamine oxidases. Cells with high FAD autofluorescence were mostly intact and were not co-labelled with indicators for necrosis or apoptosis. However, cells with high FAD fluorescence showed activation of apoptosis and necrosis within 24 h after initial measurements. Thus, high level of FAD autofluorescence is an indicator of cell pathology and reveals an upcoming apoptosis and necrosis.


Flavin-Adenine Dinucleotide , Mitochondria , Humans , Flavin-Adenine Dinucleotide/metabolism , Mitochondria/metabolism , Fibroblasts/metabolism , Cell Death , Necrosis/metabolism
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