Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate l-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an Escherichia coli expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H(2)O(2)-supported oxidation of N(omega)-hydroxy-l-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280-Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280-Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with l-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280-Ala mutant does not bind l-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity.
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PMID:Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions. 1151 17

The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group. Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-13C-FAD or 2,4a-13C-FMN. In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea. The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole. Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution. The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution. This on-column reconstitution method can also be used in cases where the apoprotein is unstable. Therefore, it may develop as a universal method for replacement of flavin or other cofactors.
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PMID:A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins. 1252 9

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous bacterial flavoenzymes that contain covalently bound flavin [8alpha-(S-cysteinyl)FAD]. Reaction of MSOX or MTOX with a small excess of sodium borohydride results in immediate flavin reduction to a species that exhibits spectral properties (lambda(max) = 405 nm with a second broad peak at 332 nm) similar to those of 3,4-dihydroflavin. The borohydride-reduced enzymes retain full catalytic activity. Substrate reduction converts the 405 nm species to an air-sensitive tetrahydroflavin that reacts with oxygen to yield unmodified oxidized enzyme. Unexpectedly, the putative 3,4-dihydroflavin bound to MSOX or MTOX is unstable in the absence of substrate. An isosbestic conversion of the 405 nm species to yield unmodified, oxidized flavin is observed when the reaction is conducted under aerobic conditions (k(obs) = 4.9 x 10(-2) min(-1)). Under anaerobic conditions, an oxygen-sensitive species resembling 1,5-dihydroflavin is formed in an isosbestic reaction that occurs at a rate similar to that of the aerobic reaction (k(obs) = 5.3 x 10(-2) min(-1)). Possible reaction of the 3,4-dihydroflavin with a second molecule of borohydride to yield an air-sensitive tetrahydroflavin is unlikely since prior scavenging of residual borohydride with excess formaldehyde had no effect on the aerobic conversion to unmodified oxidized flavin. The observed instability is attributed to a tautomeric rearrangement of the 3,4-dihydroflavin to generate 1,5-dihydroflavin, a species that is also air-sensitive. Evidence in favor of an active site facilitated tautomerization reaction is provided by the fact that the stability of the 405 nm species formed with MSOX is enhanced 200-fold upon denaturation with urea or heat. The observed tautomeric rearrangement of 3,4-dihydroflavin may provide insight regarding a related flavin tautomerization reaction that has been proposed as a key step in the biosynthesis of covalent flavin linkages.
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PMID:Tautomeric rearrangement of a dihydroflavin bound to monomeric sarcosine oxidase or N-methyltryptophan oxidase. 1254 3

Impaired functioning of methylenetetrahydrofolate reductase (MTHFR) can cause high levels of homocysteine in plasma or hyperhomocysteinemia, which is an independent risk factor for cardiovascular diseases and neural tube defects. We have studied in detail the effect of modulation of hydrophobic and electrostatic interactions of Escherichia coli MTHFR on its structure and function. Alterations in hydrophobic interactions of MTHFR, using urea, lead to dissociation of the native tetramer, resulting in stabilization of enzymatically active holoenzyme dimers followed by unfolding of the holoenzyme dimer to the denatured monomer along with dissociation of FAD from the enzyme. This is the first report of an enzymatically active dimer of E. coli MTHFR and suggests that the dimer rather than tetramer is the smallest functionally active unit of the enzyme. Furthermore, these results also demonstrate that dissociation of the FAD cofactor from the enzyme occurs only on unfolding of the dimer to denatured monomers. Modulation of electrostatic interactions, using NaCl, leads to dissociation of the native enzyme, resulting in stabilization of an enzymatically inactive partially unfolded holoenzyme dimer. Comparative analysis of loss of enzymatic activity and changes in structural features of MTHFR demonstrate a very good correlation between enhanced flexibility of the enzyme-bound FAD and loss of enzymatic activity, suggesting the importance of rigidity of the FAD cofactor in maintenance of the enzymatic activity of MTHFR.
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PMID:Unique holoenzyme dimers of the tetrameric enzyme Escherichia coli methylenetetrahydrofolate reductase: characterization of structural features associated with modulation of the enzyme's function. 1266 83

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.
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PMID:Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase. 1510 41

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His(69) residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (approximately 3 versus approximately 5 M, respectively), and the mutant protein had a melting temperature approximately 10-15 degrees C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 m urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.
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PMID:Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin. 1581 48

Aryl-alcohol oxidase (AAO), a flavoenzyme with unique spectral and catalytic properties that provides H2O2 for fungal degradation of lignin, has been successfully activated in vitro after Escherichia coli expression. The recombinant AAO (AAO*) protein was recovered from inclusion bodies of E. coli W3110 transformed with pFLAG1 containing the aao cDNA from Pleurotus eryngii. Optimization of in vitro refolding yielded 75% active enzyme after incubation of AAO* protein (10 microg/ml) for 80 h (at 16 degrees C and pH 9) in the presence of glycerol (35%), urea (0.6 M), glutathione (GSSG/GSH molar ratio of 2), and FAD (0.08 mM). For large-scale production, the refolding volume was 15-fold reduced and over 45 mg of pure active AAO* was obtained per liter of E. coli culture after a single anion-exchange chromatographic step. Correct FAD binding and enzyme conformation were verified by UV-visible spectroscopy and circular dichroism. Although the three enzymes oxidized the same aromatic and aliphatic polyunsaturated primary alcohols, some differences in physicochemical properties, including lower pH and thermal stability, were observed when the activated enzyme was compared with fungal AAO from P. eryngii (wild enzyme) and Emericella nidulans (recombinant enzyme), which are probably related to the absence of glycosylation in the E. coli expressed AAO.
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PMID:In vitro activation, purification, and characterization of Escherichia coli expressed aryl-alcohol oxidase, a unique H2O2-producing enzyme. 1603 72

The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild-type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild-type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo- and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea-induced unfolding does not occur by a simple two-state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.
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PMID:Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD. 1821 20

The up-regulation of transcobalamins [hitherto posited as indicating a central need for cobalamin (Cbl) in inflammation], whose expression, like inducible nitric oxide synthase (iNOS), is Sp1- and interferondependent, together with increased intracellular formation of glutathionylcobalamin (GSCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), may be essential for the timely promotion and later selective inhibition of iNOS and concordant regulation of endothelial and neuronal NOS (eNOS/nNOS.) Cbl may ensure controlled high output of nitric oxide (NO) and its safe deployment, because: (1) Cbl is ultimately responsible for the synthesis or availability of the NOS substrates and cofactors heme, arginine, BH(4) flavin adenine dinucleotide/flavin mononucleotide (FAD/FMN) and NADPH, via the far-reaching effects of the two Cbl coenzymes, methionine synthase (MS) and methylmalonyl CoA mutase (MCoAM) in, or on, the folate, glutathione, tricarboxylic acid (TCA) and urea cycles, oxidative phosphorylation, glycolysis and the pentose phosphate pathway. Deficiency of any of theNOS substrates and cofactors results in 'uncoupled' NOS reactions, decreasedNO production and increased or excessive O(2) (-), H(2)O(2), ONOO(-) and other reactive oxygen species (ROS), reactive nitric oxide species (RNIS) leading to pathology. (2) Cbl is also the overlooked ultimate determinant of positive glutathione status, which favours the formation of more benign NO species, s-nitrosothiols, the predominant form in which NO is safely deployed. Cbl status may consequently act as a 'back-up disc' that ensures the active status of antioxidant systems, as well as reversing and modulating the effects of nitrosylation in cell signal transduction.New evidence shows that GSCbl can significantly promote iNOS/ eNOS NO synthesis in the early stages of inflammation, thus lowering high levels of tumour necrosis factor-a that normally result in pathology, while existing evidence shows that in extreme nitrosative and oxidative stress, GSCbl can regenerate the activity of enzymes important for eventual resolution, such as glucose 6 phosphate dehydrogenase, which ensures NADPH supply, lactate dehydrogenase, and more; with human clinical case studies of OHCbl for cyanide poisoning, suggesting Cbl may regenerate aconitase and cytochrome c oxidase in the TCA cycle and oxidative phosphorylation. Thus, Cbl may simultaneously promote a strong inflammatory response and the means to resolve it.
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PMID:The return of the Scarlet Pimpernel: cobalamin in inflammation II - cobalamins can both selectively promote all three nitric oxide synthases (NOS), particularly iNOS and eNOS, and, as needed, selectively inhibit iNOS and nNOS. 1883 33

Usually during the folding/unfolding of flavoproteins, an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations, were found to interact selectively with the NADP+-binding domain of TgFNR and to induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.
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PMID:Guanidine hydrochloride- and urea-induced unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase: stabilization of a functionally inactive holo-intermediate. 1923 41


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