ABSTRACT
Enzyme-mediator bioconjugation is emerging as a building block for designing electrode platforms for the construction of biosensors and biofuel cells. Here, we report a one-pot bioconjugation technique for flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and thionine (TH) using a series of cross-linkers, including epoxy, N-hydroxysuccinimide (NHS), and aldehydes. In this technique, FAD-GDH and thionine are conjugated through an amine cross-linking reaction to generate a redox network, which has been successfully employed for the oxidation of glucose. The bioconjugation chemistry of cross-linkers with the amino groups on FAD-GDH and thionine plays a vital role in generating distinct network structures. The epoxy-type cross-linker reacts with the primary and secondary amines of thionine at room temperature, thereby producing an FAD-GDH-TH-FAD-GDH hyperbranched bioconjugate network, the aldehyde undergoes a rapid cross-linking reaction to produce a network of FAD-GDH-FAD-GDH, while the NHS-based cross-linker can react with the primary amines of both FAD-GDH and thionine, forming an FAD-GDH-cross-linker-TH polymeric network. This reaction has the potential to enable the conjugation of a redox mediator with a FAD-GDH network, which is particularly essential when designing an enzyme electrode platform. The data demonstrated that the polymeric cross-linked network based on the NHS cross-linker exhibited a considerable increase in electron transport while producing a catalytic current of 830 µA cm-2. The cross-linker spacer arm length also affects the overall electrochemical function of the network and its performance; an adequate spacer length containing a cross-linker is required, resulting in a faster electron transfer. Finally, a leaching test confirmed that the stability of the enzyme electrode was improved when the electrode was tested using the redox probe. This study elucidates the relationship between cross-linking chemistry and redox network structure and enhances the high performance of enzyme electrode platforms for the oxidation of glucose.
Subject(s)
Biosensing Techniques , Cross-Linking Reagents , Glucose 1-Dehydrogenase , Oxidation-Reduction , Phenothiazines , Phenothiazines/chemistry , Glucose 1-Dehydrogenase/chemistry , Glucose 1-Dehydrogenase/metabolism , Cross-Linking Reagents/chemistry , Biosensing Techniques/methods , Glucose/chemistry , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Electrodes , Electrochemical Techniques , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , BiocatalysisABSTRACT
Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2-12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.
Subject(s)
Enzyme Stability , FMN Reductase , Geobacillus , Geobacillus/enzymology , Geobacillus/genetics , FMN Reductase/genetics , FMN Reductase/metabolism , FMN Reductase/chemistry , Cloning, Molecular , Hydrogen-Ion Concentration , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/isolation & purification , Amino Acid Sequence , Kinetics , Molecular Docking Simulation , Temperature , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Models, Molecular , Binding Sites , Escherichia coli/genetics , Mixed Function OxygenasesABSTRACT
OBJECTIVE: The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. METHODS: The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. RESULTS: OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. CONCLUSIONS: Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.
Subject(s)
B-Lymphocytes , Flavin-Adenine Dinucleotide , Flow Cytometry , NAD , Single-Cell Analysis , Humans , Flow Cytometry/methods , NAD/metabolism , Flavin-Adenine Dinucleotide/metabolism , Single-Cell Analysis/methods , B-Lymphocytes/metabolism , Mitochondria/metabolism , T-Lymphocytes/metabolism , Oxidation-Reduction , Fluorescence , Arthritis, Rheumatoid/metabolism , Glycolysis , Oxidative Phosphorylation , Female , Male , Glucose/metabolismABSTRACT
Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Optical Imaging , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/pathology , Carcinoma, Hepatocellular/diagnostic imaging , Carcinoma, Hepatocellular/pathology , Optical Imaging/methods , Humans , Animals , Neoplasm Metastasis , Biopsy , Mice , NAD/metabolism , Photons , Flavin-Adenine Dinucleotide/metabolism , Cell Line, TumorABSTRACT
Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCE: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.
Subject(s)
Mycobacterium smegmatis , Mycobacterium tuberculosis , Riboflavin , Riboflavin/biosynthesis , Riboflavin/metabolism , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Humans , Flavin-Adenine Dinucleotide/metabolism , Biosynthetic Pathways/genetics , Gene Knockdown Techniques , Mucosal-Associated Invariant T Cells/metabolism , Gene Expression Regulation, BacterialABSTRACT
Cryptochromes are essential flavoproteins for circadian rhythms and avian magnetoreception. Flavin adenine dinucleotide (FAD), a chromophore within cryptochromes, absorbs blue light, initiating electron transfer processes that lead to a biological signaling cascade. A key step in this cascade is the formation of the FAD semiquinone radical (FADHâ¢), characterized through a specific red-light absorption. The absorption spectra of FADH⢠in cryptochromes are, however, significantly different from those recorded for the cofactor in solution, primarily due to protein-induced shifts in the absorption peaks. This study employs a multiscale approach, combining molecular dynamics (MD) simulations with quantum mechanical/molecular mechanical (QM/MM) methodologies, to investigate the influence of protein dynamics on embedded FADH⢠absorption. We emphasize the role of the protein's polarizable environment in the shaping of the absorption spectrum, crucial for accurate spectral predictions in cryptochromes. Our findings provide valuable insights into the absorption process, advancing our understanding of cryptochrome functioning.
Subject(s)
Arabidopsis , Cryptochromes , Flavin-Adenine Dinucleotide , Molecular Dynamics Simulation , Quantum Theory , Cryptochromes/chemistry , Cryptochromes/metabolism , Arabidopsis/metabolism , Arabidopsis/chemistry , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolismABSTRACT
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.
Subject(s)
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/chemistryABSTRACT
BACKGROUND: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. METHODS: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). RESULTS: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M- 1.S- 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M- 1.S- 1. CONCLUSIONS: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.
Subject(s)
Flavin Mononucleotide , Flavin-Adenine Dinucleotide , Riboflavin , Schistosoma mansoni , Riboflavin/metabolism , Flavin Mononucleotide/metabolism , Animals , Flavin-Adenine Dinucleotide/metabolism , Schistosoma mansoni/metabolism , Schistosoma mansoni/genetics , Mice , Humans , Schistosomiasis mansoni/parasitology , Schistosomiasis mansoni/metabolismABSTRACT
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.
Subject(s)
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 BindingABSTRACT
Enzymes play a fundamental role in cellular metabolism. A wide range of enzymes require the presence of complementary coenzymes and cofactors to function properly. While coenzymes are believed to have been part of the last universal ancestor (LUCA) or have been present even earlier, the syntheses of crucial coenzymes like the redox-active coenzymes flavin adenine dinucleotide (FAD) or nicotinamide adenine dinucleotide (NAD+) remain challenging. Here, we present a pathway to NAD+ under prebiotic conditions starting with ammonia, cyanoacetaldehyde, prop-2-ynal and sugar-forming precursors, yielding inâ situ the nicotinamide riboside. Regioselective phosphorylation and water stable light activated adenosine monophosphate derivatives allow for topographically and irradiation-controlled formation of NAD+. Our findings indicate that NAD+, a coenzyme vital to life, can be formed non-enzymatically from simple organic feedstock molecules via photocatalytic activation under prebiotically plausible early Earth conditions in a continuous process under aqueous conditions.
Subject(s)
NAD , NAD/chemistry , NAD/metabolism , Ammonia/chemistry , Niacinamide/chemistry , Niacinamide/analogs & derivatives , Phosphorylation , Prebiotics , Adenosine Monophosphate/chemistry , Catalysis , Acetaldehyde/chemistry , Oxidation-Reduction , Water/chemistry , Pyridinium Compounds/chemistry , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolismABSTRACT
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 (FADHâ¢) 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.
Subject(s)
Cryptochromes , Flavin-Adenine Dinucleotide , Cryptochromes/chemistry , Cryptochromes/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Animals , Avian Proteins/chemistry , Avian Proteins/metabolism , Oxidation-ReductionABSTRACT
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.
Subject(s)
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, MolecularABSTRACT
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.
Subject(s)
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 TransportABSTRACT
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 â¼20 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.
Subject(s)
Dihydroorotate Dehydrogenase , Lactococcus lactis , Biocatalysis , Catalysis , Catalytic Domain , Flavin Mononucleotide/metabolism , Flavin Mononucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Kinetics , Lactococcus lactis/enzymology , Lactococcus lactis/metabolism , NAD/metabolism , NAD/chemistry , Oxidation-ReductionABSTRACT
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.
Subject(s)
Calcium , NADPH Oxidase 5 , NADP , Humans , Binding Sites , Calcium/metabolism , Cryoelectron Microscopy , Electron Transport , Enzyme Activation , Flavin-Adenine Dinucleotide/metabolism , Molecular Dynamics Simulation , NADP/metabolism , NADPH Oxidase 5/metabolism , NADPH Oxidase 5/genetics , NADPH Oxidase 5/chemistry , Protein Binding , Reactive Oxygen Species/metabolism , Superoxides/metabolism , Zinc/metabolismABSTRACT
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.
Subject(s)
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/metabolismABSTRACT
Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity.
Subject(s)
Catalytic Domain , Flavin-Adenine Dinucleotide , Models, Molecular , Protein Binding , Humans , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Crystallography, X-Ray , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/genetics , Protein Multimerization , Binding Sites , Protein Domains , Amino Acid SequenceABSTRACT
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.
Subject(s)
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 TheoryABSTRACT
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.
Subject(s)
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/metabolismABSTRACT
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.