Coenzyme FAD Research Articles

Modelling the elusive conformational activation in kynurenine 3-monooxygenase.

Kynurenine 3-monooxygenase (KMO) regulates the levels of important physiological intermediates in the kynurenine pathway [Guillemin, et al., Journal of Neuroscience, 2007, 27, 12884], which is the major route for L-tryptophan catabolism. Its catalytic activity (hydroxylation) is dependent on the formation of a short-lived intermediate that forms after the reduction of the coenzyme FAD. The reduction takes place fast when the substrate binds to KMO. Crystal structures of the apo form and in complex with an effector inhibitor, which prevents the hydroxylation of the substrate but also stimulates KMO like the substrate, and a competitive inhibitor, which suppresses the substrate hydroxylation, are available for the resting in conformation only. The active out conformational state that enables the reduction of FAD at an exposed location of KMO after its stimulation by an effector, however, was implicated but not resolved experimentally and has remained elusive so far. Molecular dynamics simulations of apo KMO and the inhibitor-KMO complexes are carried out using extensive multi-dimensional umbrella sampling to explore the free-energy surface of the coenzyme FAD's conformational conversion from the in state (buried within the active site) to the out state. This allows a discussion and comparison with the experimental results, which showed a significant increase in the rate of reduction of FAD in the presence of an effector inhibitor and absence of enzymatic function in the presence of a competitive inhibitor [Kim, et al., Cell Chemical Biology, 2018, 25, 426]. The free-energy barriers associated with those conformational changes and structural models for the active out conformation are obtained. The interactions during the conformational changes are determined to identify the influence of the effector.

Machine-learning model selection and parameter estimation from kinetic data of complex first-order reaction systems.

Dealing with a system of first-order reactions is a recurrent issue in chemometrics, especially in the analysis of data obtained by spectroscopic methods applied on complex biological systems. We argue that global multiexponential fitting, the still common way to solve such problems, has serious weaknesses compared to contemporary methods of sparse modeling. Combining the advantages of group lasso and elastic net-the statistical methods proven to be very powerful in other areas-we created an optimization problem tunable from very sparse to very dense distribution over a large pre-defined grid of time constants, fitting both simulated and experimental multiwavelength spectroscopic data with high computational efficiency. We found that the optimal values of the tuning hyperparameters can be selected by a machine-learning algorithm based on a Bayesian optimization procedure, utilizing widely used or novel versions of cross-validation. The derived algorithm accurately recovered the true sparse kinetic parameters of an extremely complex simulated model of the bacteriorhodopsin photocycle, as well as the wide peak of hypothetical distributed kinetics in the presence of different noise levels. It also performed well in the analysis of the ultrafast experimental fluorescence kinetics data detected on the coenzyme FAD in a very wide logarithmic time window. We conclude that the primary application of the presented algorithms-implemented in available software-covers a wide area of studies on light-induced physical, chemical, and biological processes carried out with different spectroscopic methods. The demand for this kind of analysis is expected to soar due to the emerging ultrafast multidimensional infrared and electronic spectroscopic techniques that provide very large and complex datasets. In addition, simulations based on our methods could help in designing the technical parameters of future experiments for the verification of particular hypothetical models.

Decay Times and Anisotropy in Polarized Fluorescence of Flavin Adenine Dinucleotide Determined with Subnanosecond Resolution

Optical properties of biological coenzyme FAD in an aqueous solution have been studied. Two fluorescence lifetimes, a rotational diffusion time, and an anisotropy parameter have been determined from experiments by recording the decay of polarized fluorescence excited by picosecond laser pulses. The results obtained are discussed and compared with the results of other authors.

Fluorescence spectral detection of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML): A novel photodiagnosis strategy.

Acute lymphoblastic leukemia (ALL) is a cancer of the lymphoid line of blood cell, showing a rapid growth of lymphoblastic immature cells. Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells. Early diagnosis is crucial for the effective treatment of these patients. The current standard procedure of diagnosis is extensive blood count analysis, microscopic morphological investigations, bone marrow biopsy and flow-cytometer which are all time- consuming and expensive. Here we demonstrate the advantage a new technique for the diagnosis of ALL and AML based on the fluorescence spectra of blood plasma and RBCs samples from the above patients. Based on the 85 patients analyzed the results reveal that our approach could discriminate the two malignancies unambiguously from the normal with 88 % sensitivity and 80 % specificity. The abnormal decrease in the level of amino acid, tryptophan and coenzyme NADH and abnormal increase in the other amino acid, tyrosine and another coenzyme FAD, (both in comparison to the normal control) act as malignancy indicative biomarkers. Since the contrast parameter between the normal and malignant samples is four- fold, the potential for early detection of leukemia is high. The instrumentation and technique are new and simple; hence may have significant supplementary or complementary value with the existing diagnostic methods.

Comprehensive Profile of Acute Mitochondrial Dysfunction in a Preclinical Model of Severe Penetrating TBI.

Mitochondria constitute a central role in brain energy metabolism, and play a pivotal role in the development of secondary pathophysiology and subsequent neuronal cell death following traumatic brain injury (TBI). Under normal circumstances, the brain consumes glucose as the preferred energy source for adenosine triphosphate (ATP) production over ketones. To understand the comprehensive picture of substrate-specific mitochondrial bioenergetics responses following TBI, adult male rats were subjected to either 10% unilateral penetrating ballistic-like brain injury (PBBI) or sham craniectomy (n = 5 animals per group). At 24 h post-injury, mitochondria were isolated from pooled brain regions (frontal cortex and striatum) of the ipsilateral hemisphere. Mitochondrial bioenergetics parameters were measured ex vivo in the presence of four sets of metabolic substrates: pyruvate+malate (PM), glutamate+malate (GM), succinate (Succ), and β-hydroxybutyrate+malate (BHBM). Additionally, mitochondrial matrix dehydrogenase activities [i.e., pyruvate dehydrogenase complex (PDHC), alpha-ketoglutarate dehydrogenase complex (α-KGDHC), and glutamate dehydrogenase (GDH)] and mitochondrial membrane-bound dehydrogenase activities [i.e., electron transport chain (ETC) Complex I, II, and IV] were compared between PBBI and sham groups. Furthermore, mitochondrial coenzyme contents, including NAD(t) and FAD(t), were quantitatively measured in both groups. Collectively, PBBI led to an overall significant decline in the ATP synthesis rates (43–50%; *p < 0.05 vs. sham) when measured using each of the four sets of substrates. The PDHC and GDH activities were significantly reduced in the PBBI group (42–53%; *p < 0.05 vs. sham), whereas no significant differences were noted in α-KGDHC activity between groups. Both Complex I and Complex IV activities were significantly reduced following PBBI (47–81%; *p < 0.05 vs. sham), whereas, Complex II activity was comparable between groups. The NAD(t) and FAD(t) contents were significantly decreased in the PBBI group (27–35%; *p < 0.05 vs. sham). The decreased ATP synthesis rates may be due to the significant reductions in brain mitochondrial dehydrogenase activities and coenzyme contents observed acutely following PBBI. These results provide a basis for the use of “alternative biofuels” for achieving higher ATP production following severe penetrating brain trauma.

Калікс[4]арени як модулятори енергозалежної акумуляції Са2+ та функціонування електронно-транспортного ланцюга в мітохондріях гладенького м’яза

The influence of supramolecular macrocyclic compounds calix[4]arenes (C-97, C-99, C-107) at a concentration of 100 nM in the process of energy-dependent Ca²⁺-transport in isolated mitochondria of smooth muscle, as well as autofluorescence mitochondrial coenzyme NADH, FAD and hydrodynamic diameter of these organelles was investigated. Using Ca²⁺-sensitive fluorescent dye Fluo-4 AM it was shown that the selected calix[4]arenes can suppress energy-dependent accumulation of Ca²⁺ by mitochondria. Accumulation of Ca²⁺ (80 jiM in the medium) accompanied by the growth of the fluorescent probe response from a conventional unit to a value of 1,57±0,04 (n=5). Calix[4]arenes C-97, C-99, C-107 falls fluorescent signal below the 0,88±0,08, 0,92±0,08 and 0,78±0,04 respectively. Thus, the selected calix[4]arenas lead to release of previously accumulated Ca2+ from mitochondria. Under the influence of C-97 and C-99 fluorescent signal from NADH reduced to -0,11±0,02 and -0,12±0,02, respectively, in relation to the reference value - -0,05±0,01 (n=5). Analysis of fluorescence response NADH and FAD in a suspension of isolated mitochondria suggests that the effects of test compounds on the functional activity of the electron transport chain is associated with the initial stimulation of its 1-th complex and subsequent inhibition of Ca²⁺-dependent NAD- containing Krebs cycle dehydrogenases. Along with this, the use of photon correlation spectroscopy to assess changes in the volume of mitochondria (their hydrodynamic diameter) under the action of selected calix[4]arenes has shown that interference with the electron transport chain leads to changes in the osmotic balance between the matrix of the mitochondria and the external environment. The result is the growth of isolated organelles volume. In particular, the hydrodynamic diameter of mitochondria increased by 22±6 % and 34±8 % (n=5) in presence of C-97 or C-99. The conclusion was done about the advisability of further studies of the calyx[4]arenes effect on smooth muscle Ca²⁺-homeostase and mitochondrial bioenergetics in order to find effective modifiers of their func- tional activity.

The catalytic mechanism of decarboxylative hydroxylation of salicylate hydroxylase revealed by crystal structure analysis at 2.5 Å resolution

The X-ray crystal structure of a salicylate hydroxylase from Pseudomonas putida S-1 complexed with coenzyme FAD has been determined to a resolution of 2.5 Å. Structural conservation with p- or m-hydroxybenzoate hydroxylase is very good throughout the topology, despite a low amino sequence identity of 20–40% between these three hydroxylases. Salicylate hydroxylase is composed of three distinct domains and includes FAD between domains I and II, which is accessible to solvent. In this study, which analyzes the tertiary structure of the enzyme, the unique reaction of salicylate, i.e. decarboxylative hydroxylation, and the structural roles of amino acids surrounding the substrate, are considered.

Spectrally matched upconverting luminescent nanoparticles for monitoring enzymatic reactions.

We report on upconverting luminescent nanoparticles (UCLNPs) that are spectrally tuned such that their emission matches the absorption bands of the two most important species associated with enzymatic redox reactions. The core-shell UCLNPs consist of a β-NaYF4 core doped with Yb(3+)/Tm(3+) ions and a shell of pure β-NaYF4. Upon 980 nm excitation, they display emission bands peaking at 360 and 475 nm, which is a perfect match to the absorption bands of the enzyme cosubstrate NADH and the coenzyme FAD, respectively. By exploiting these spectral overlaps, we have designed fluorescent detection schemes for NADH and FAD that are based on the modulation of the emission intensities of UCLNPs by FAD and NADH via an inner filter effect.

Characterization of coenzyme binding and selectivity determinants in Mycobacterium tuberculosis flavoprotein reductase A: analysis of Arg199 and Arg200 mutants at the NADP(H) 2′-phosphate binding site

Mycobacterium tuberculosis FprA (flavoprotein reductase A) is an NAD(P)H- and FAD-binding reductase that is structurally/evolutionarily related to adrenodoxin reductase. Structural analysis implicates Arg(199) and Arg(200) in interactions with the NADP(H) 2'-phosphate group. R199A, R200A and R199A/R200A mutants were characterized to explore the roles of these basic residues. All mutations abolished neutral FAD semiquinone stabilization in the NADPH-reduced enzyme, owing to weakened NADPH affinity. Instead, FAD hydroquinone was formed in all mutants, and each displayed substantially enhanced autooxidation rates (20-40-fold) compared with NADPH-reduced WT (wild-type) FprA. Steady-state ferricyanide reduction studies revealed diminished NADPH affinity (higher K(m) values), but lower NADH K(m) values. Despite a lowered k(cat), the R199A/R200A mutant exhibited a 200-fold coenzyme specificity switch towards NADH, although substrate inhibition was observed at high NADH concentrations (K(i)=250 microM). Stopped-flow FAD reduction studies confirmed substantially increased NADPH K(d) values, although the limiting flavin reduction rate constant was similar in all mutants. The R199A mutation abolished electron transfer between hydroquinone FprA and NADP+, while this reaction progressed (via an FADH(2)-NADP+ charge-transfer intermediate) for R200A FprA, albeit more slowly (k(lim)=58.1 s(-1) compared with >300 s(-1)) than in WT. All mutations caused positive shifts in FAD potential (approximately 40-65 mV). Binding of an NADPH analogue (tetrahydro-NADP) induced negative shifts in potential ( approximately 30-40 mV) only for variants with the R200A mutation, indicating distinctive effects of Arg(199)/Arg(200) on coenzyme binding mode and FAD potential. Collectively, these data reveal important roles for the phylogenetically conserved arginines in controlling FprA FAD environment, thermodynamics, coenzyme selectivity and reactivity.

The Proline-Dependent Transcription Factor Put3 Regulates the Expression of the Riboflavin Transporter MCH5 in Saccharomyces cerevisiae

Like most microorganisms, the yeast Saccharomyces cerevisiae is prototrophic for riboflavin (vitamin B2). Riboflavin auxotrophic mutants with deletions in any of the RIB genes frequently segregate colonies with improved growth. We demonstrate by reporter assays and Western blots that these suppressor mutants overexpress the plasma-membrane riboflavin transporter MCH5. Frequently, this overexpression is mediated by the transcription factor Put3, which also regulates the proline catabolic genes PUT1 and PUT2. The increased expression of MCH5 may increase the concentrations of FAD, which is the coenzyme required for the activity of proline oxidase, encoded by PUT1. Thus, Put3 regulates proline oxidase activity by synchronizing the biosynthesis of the apoenzyme and the coenzyme FAD. Put3 is known to bind to the promoters of PUT1 and PUT2 constitutively, and we demonstrate by gel-shift assays that it also binds to the promoter of MCH5. Put3-mediated transcriptional activation requires proline as an inducer. We find that the increased activity of Put3 in one of the suppressor mutants is caused by increased intracellular levels of proline. Alternative PUT3-dependent and -independent mechanisms might operate in other suppressed strains.

Detection Mechanism of Metallized Carbon Epoxy Oxidase Enzyme Based Sensors

AbstractThe coenzyme FAD has been identified to play an important role in the detection mechanism of oxidase enzyme based biosensors. Incorporating FAD into the carbon composite improved sensitivity to H2O2 consequently increasing sensitivity to the respective analyte. The amount of active enzyme also increased thus enhancing the overall performance of the sensors. Polycarboxybetaine (PCB) has been used as a biocompatible membrane coating. The PCB coated sensors gave reproducible calibrations in protein solutions, which has been shown to be a valid protocol for testing biocompatibility. The importance of reporting selectivity in a manner which indicates the “fitness of purpose” of biosensors has been discussed.

A Tetrameric Model of the Dihydrolipoamide Dehydrogenase Component of the Pyruvate Dehydrogenase Complex: Construction and Evaluation by Molecular Modeling Techniques

On the basis of the homodimeric X-ray structure of dihydrolipoamide dehydrogenase from Azotobacter vinelandii we demonstrate by protein modeling techniques that two dimeric units of this enzyme can associate to a tetrameric structure with intense contacts between the building blocks. Complementary structures of the respective other unit in the tetramer contribute to the active sites. The coenzyme FAD becomes shielded from the environment, thus its binding is stabilized. By energy minimization techniques binding energies and RMS-values were computed and the contact areas between the building blocks were determined to quantify the interaction. In the cell tetramerization of dihydrolipoamide dehydrogenase will be realized upon its incorporation as an enzyme component into the pyruvate dehydrogenase multienzyme complex and will have consequences for the structure and subunit stoichiometry of the complex. Especially, the multiplicity of the three enzyme components, i.e. pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase in the enzyme complex must be 24:24:24 instead of 24:24:12 assumed so far.

Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.

Stability and Kinetic Properties of Immobilized Rhodotorula gracilisd‐Amino Acid Oxidase

d‐Amino acid oxidase form Rhodotorula gracilis has been immobilized with the use of amine reactive coupling chemistries. Coupling to Affi‐Gel 10 was very effective: yields of 50 to 60% were attained under optimal conditions with a specific activity of immobilized enzyme comparable to that of the free suspended preparation. No loss of the coenzyme FAD was observed under any experimental conditions. Immobilized d‐amino acid oxidase exhibited a marked enhancement of stability against inactivation on heating, on pH shifts, and on denaturating agents; a relevant operational and storage stability was furthermore shown by the coupled system. Substrate specificity of the Affi‐Gel‐coupled enzyme was broader than for free enzyme; lowered apparent Kms and unchanged specific activity values for d‐alanine, d‐leucine, d‐proline, d‐valine, and cephalosporin C were determined for immobilized enzyme compared to the soluble form.

Crystallization and preliminary X-ray diffraction studies of a deglycosylated glucose oxidase from Penicillium amagasakiense

The dimeric glucose oxidase from Penicillium amagasakiense was deglycosylated, purified and crystallized as a complex with its coenzyme FAD. Deglycosylation and purification to isoelectric homogeneity were shown to be an important prerequisite step to obtain crystals suitable for X-ray investigations. Crystals of the deglycosylated enzyme were reproducibly grown using ammonium sulfate as precipitant at pH 7.4 to 7.5. Crystals diffract to at least 2.0 Å resolution and belong to the orthorhombic space group P2 12 12 1, with refined lattice constants of a = 59.3 A ̊ , b = 136.3 A ̊ and c = 156.7 A ̊ . Assuming two monomers (≈ 135 kDa) per asymmetric unit the V m value is 2.3 Å 3/Da.

Effect of Respiratory Infection on Tissue Riboflavin and Flavin Enzymes in Mice

The effects of Klebsiella pneumoniae infection on red blood cell and liver riboflavin levels and a few flavin enzymes were examined to verify our earlier hypothesis based on human experience that upper respiratory infections result in mobilization of riboflavin from tissues into blood, resulting in a rise in red blood cell riboflavin and an increased saturation of erythrocyte glutathione reductase (EGR) with its coenzyme flavin adenine dinucleotide (FAD; reduction in EGR activation coefficient, AC, values). Thirty-six-day-old male mice fed a diet marginally sufficient in riboflavin were injected with a single sublethal dose of K. pneumoniae. Batches of control, deficient infected and deficient uninfected animals were killed during the peak period of infection and after recovery. Infection brought about a significant rise in basal EGR activity due to greater saturation with coenzyme FAD and a concomitant reduction in EGR-AC values. On recovery, basal EGR activity decreased, and EGR-AC values increased to levels beyond control values, suggesting riboflavin deficiency. In the liver, acyl-coenzyme A dehydrogenase showed a significant decrease and pyridoxamine-phosphate oxidase activity a significant increase. Both the enzymes normalized after recovery. D-amino acid oxidase activity did not change.

Mechanism of inhibition of erythrocyte glutathione reductase by Mitomycin-C

Mitomycin-C (MT-C) inhibits human erythrocyte glutathione reductase (EGR). We found that k m values of EGR for GSSG, NADPH, and FAD were 1.3 × 10 −4 mol/l, 2.3 × 10 −5 mol/l, and 3.9 × 10 −7 mol/l respectively, and that EGR was inhibited by MT-C non-competitively with respect to both the substrate GSSG ( K i = 4.8 × 10 −5 mol/l) and the cofactor NADPH ( K i = 2.2 × 10 −4 mol/l), and competitively with respect to FAD ( K i = 4.2 × 10 −5 mol/l). On the other hand, we demonstrated that FAD makes a complex with MT-C, the dissociation constant ( K = 6.5 × 10 −5 mol/l) of which was obtained from fluorescence quenching of FAD with MT-C. It also became clear that spectra of FAD at around 375 nm changed with increasing MT-C concentration. So, the data strongly suggest that the mechanism of inhibition of EGR by MT-C is mainly due to complex formation of the co-enzyme FAD with MT-C.

X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques

The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 A electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the "phased translation function", which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 A, the resolution was gradually extended to 2.8 A in another 140 cycles. The 2.8 A electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD.(ABSTRACT TRUNCATED AT 400 WORDS)

The formation of reactive intermediates in the MAO-catalyzed oxidation of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Oxidation of MPTP by monoamine oxidase (MAO), leading to the formation of reactive metabolites, is a critical step in the expression of the nigrostriatal toxicity of this molecule. A catalytic mechanism for the 2-electron oxidation of MPTP to MPDP + and for the further 2-electron oxidation of MPDP + to MPP + is proposed, involving the formation of carbon-centered radical intermediates. These radical species appear to be involved in the mechanism-based inactivation of MAO by MPTP, possibly by generating 1,4-dihydropyridine adducts with the enzyme apoprotein or its coenzyme FAD. The pathways of metabolism of MPTP in brain and peripheral tissues and the active accumulation of metabolites of MPTP in dopaminergic neurons are discussed in terms of their possible contribution to the selective cytotoxicity of the compound.