Flavin Adenine Dinucleotide Prosthetic Group Research Articles

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

C-terminal residues of ferredoxin-NAD(P)+ reductase from Chlorobaculum tepidum are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP+/NADPH.

Ferredoxin-NAD(P)+ reductase ([EC 1.18.1.2], [EC 1.18.1.3]) from Chlorobaculum tepidum (CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions of CtFNR with NADP+ and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on the re-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP+ and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326-Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-type CtFNR. In contrast, the deletions of Phe337-Glu360 and Ser338-Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region of CtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP+/NADPH.

Empirical Valence Bond Simulations of the Hydride-Transfer Step in the Monoamine Oxidase A Catalyzed Metabolism of Noradrenaline.

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines, such as dopamine, serotonin, and noradrenaline (NA), which is why they have been extensively implicated in the etiology and course of various neurodegenerative disorders and, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. The precise chemical mechanism through which MAOs regulate the amine concentration, which is vital for the development of novel inhibitors, is still not unambiguously determined in the literature. In this work, we present atomistic empirical valence bond simulations of the rate-limiting step of the MAO-A-catalyzed NA (norepinephrine) degradation, involving hydride transfer from the substrate α-methylene group to the flavin moiety of the flavin adenine dinucleotide prosthetic group, employing the full dimensionality and thermal fluctuations of the hydrated enzyme, with extensive configurational sampling. We show that MAO-A lowers the free energy of activation by 14.3 kcal mol-1 relative to that of the same reaction in aqueous solution, whereas the calculated activation free energy of ΔG‡ = 20.3 ± 1.6 kcal mol-1 is found to be in reasonable agreement with the correlated experimental value of 16.5 kcal mol-1. The results presented here strongly support the fact that both MAO-A and MAO-B isoforms function by the same hydride-transfer mechanism. We also considered a few point mutations of the "aromatic cage" tyrosine residue (Tyr444Phe, Tyr444Leu, Tyr444Trp, Tyr444His, and Tyr444Glu), and the calculated changes in the reaction barriers are in agreement with the experimental values, thus providing further support to the proposed mechanism.

Anatomy, physiology, and genetics of paragangliomas

Paragangliomas arise from an extra-adrenal paraganglionic cells, derived from the neural crest of the autonomic nervous system and make up the most common class of benign vascular neoplasms of the neck. PGLs of the head and neck originate most commonly from the paraganglia within the carotid body, the jugular foramen, the middle ear and the vagus nerve. Knowledge of the embryology, anatomy and genetics of these rare tumors is paramount to successful diagnosis and treatment of head and neck paragangliomas.

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5% of the wild type activity. The Y50W mutant retained approximately 20% reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.

Detection of intermediates in the oxidative half-reaction of the FAD-dependent thymidylate synthase from Thermotoga maritima: carbon transfer without covalent pyrimidine activation.

Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis.

Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP+ oxidoreductase from Bacillus subtilis

Ferredoxin-NADP+ oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325–K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1–10 μM. The Y313–K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin.

FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation

Renalase is a recently discovered flavoprotein that regulates blood pressure, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O 2-dependent or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 Å resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann-fold-based flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel β-sheet. A large cavity (228 Å 3), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an ‘aromatic cage’ for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k cat but higher k cat/ K m for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD + or NADP + with K d values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action.

Structure of fumarate reductase from Wolinella succinogenes at 2.2 A resolution.

Fumarate reductase couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalysed by the related complex II of the respiratory chain (succinate dehydrogenase). Here we describe the crystal structure at 2.2 A resolution of the three protein subunits containing fumarate reductase from the anaerobic bacterium Wolinella succinogenes. Subunit A contains the site of fumarate reduction and a covalently bound flavin adenine dinucleotide prosthetic group. Subunit B contains three iron-sulphur centres. The menaquinol-oxidizing subunit C consists of five membrane-spanning, primarily helical segments and binds two haem b molecules. On the basis of the structure, we propose a pathway of electron transfer from the dihaem cytochrome b to the site of fumarate reduction and a mechanism of fumarate reduction. The relative orientations of the soluble and membrane-embedded subunits of succinate:quinone oxidoreductases appear to be unique.

Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling.

The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.

A signal transducer for aerotaxis in Escherichia coli.

The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.

A Site-Directed Mutagenesis Study at Lys-113 of NAD(P)H:Quinone-Acceptor Oxidoreductase: An Involvement of Lys-113 in the Binding of the Flavin Adenine Dinucleotide Prosthetic Group

NAD(P)H: quinone-acceptor oxidoreductase (EC 1.6.99.2), also referred to as DT-diaphorase, is a flavo-protein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. Using an Escherichia coli expression system developed previously, we prepared three mutants of the rat liver quinone reductase. These mutants are Lys-113-His (K113H), Lys-113-Asp (K113D), and Lys-113-Ala (K113A). While the mutant H113H was readily purified using the same procedure as for the purification of the wild-type quinone reductase and found to have an activity similar to that of the wild-type enzyme, K113D and K113A were purified only in very small quantities, mainly in the form of apoprotein, and had very low activities. The results suggest that a positively charged amino acid at this position is important for the binding of the flavin adenine dinucleotide (FAD) prosthetic group. Flavin spectral studies of 6-mercapto-FAD-reconstituted mutants revealed that mutation at Lys-113 affects the protein environment around position-6 of the isoalloxazine ring.

52] Salicylate hydroxylase

Publisher Summary This chapter provides information on enzyme salicylate hydroxylase. Salicylate hydroxylase is a flavoprotein hydroxylase, a member of a class of enzymes elaborated by some soil bacteria, typically pseudomonads. These enzymes are induced by the presence of phenolic compounds. The activity of the enzyme was determined spectrophotometrically by following the rate of substrate-dependent oxidation of NADH in a Cary 14 or Gilford 2400 spectrophotometer at 340 nm. Salicylate hydroxylate is established as a flavoprotein containing flavin adenine dinucleotide (FAD) as the sole prosthetic group. The purified hydroxylase migrates as one homogeneous and symmetrical peak in the Model E analytical untracentrifuge with 0.1 M KCl as solvent. Enzyme activity can also be assayed by measuring the rate of oxygen consumption using an oxygen electrode. This enzyme, in addition to its FAD prosthetic group, has three substrates: salicylate or other aromatic compound, pyridine nucleotide, and oxygen. Its kinetics, therefore, can be expected to be complex.

D-Amino Acid Dehydrogenases of Pseudomonas fluorescens

Abstract Two distinct d-amino acid dehydrogenases, each showing absolute specificity for methylene blue or 2,6-dichloroindophenol, respectively, were isolated from Pseudomonas fluorescens (ATCC 11299B). The methylene blue-specific d-amino acid dehydrogenase was detectable only in extracts from d-tryptophan-grown cells and was purified about 40-fold. The 2,6-dichloroindophenol-specific d-amino acid dehydrogenase is constitutive, being present in all cell extracts irrespective of culture conditions, and it was purified about 65-fold. These enzymes appear to be flavoproteins in which the flavin adenine dinucleotide prosthetic group is tightly bound to the enzyme protein. The optimal pH of two enzymes was 7 to 8 and the Michaelis-Menten constant was 3 to 5 x 10-4 m for each d-amino acid. Substrate specificity and thermal stability were observed to be somewhat different for the two enzymes. No inhibition of these enzymes was observed with metal chelating agents.