Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.
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PMID:Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants. 191 43

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

Hydrogen peroxide reacts with two-electron reduced glutathione reductase (GR EH2 species) to give the native oxidized enzyme (E) without detectable intermediates. Prior alkylation of the EH2 interchange thiol with iodoacetamide, however, dramatically changes both the course and overall rate of the peroxide reaction. This oxidation, monitored spectrally, is characterized by an intermediate (EHRint) with enhanced long wavelength absorbance extending to 800 nm. This species decays in a second peroxide-dependent phase to an enzyme form (EHRox) easily distinguished from E. Quenching experiments with catalase allow the isolation of a stable mixture consisting of 36% monoalkylated GR (EHR), 60% EHRint, and 4% EHRox; NADPH titration and anaerobic dithiothreitol addition lead to quantitative reduction of EHRint to EHR, and there is an increase in thiol titer of 0.8-SH/FAD on NADPH reduction. Of the four titratable thiols present in EHR, 2.7 are lost on oxidation to EHRox and 0.7-0.8 mol of cysteic acid/FAD is formed. On the basis of these and other observations, we conclude that alkylation of the EH2 interchange thiol, which blocks disulfide formation, allows peroxide reaction at the remaining charge-transfer thiol to proceed via a stabilized cysteine-sulfenic acid intermediate (EHRint), which undergoes further oxidation to the corresponding cysteic acid (EHRox).
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PMID:Peroxide modification of monoalkylated glutathione reductase. Stabilization of an active-site cysteine-sulfenic acid. 191 50

3-Hydroxyphenylacetate 6-hydroxylase was purified 70-fold from a Flavobacterium sp. grown upon phenylacetic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme. SDS/PAGE of purified enzyme reveals a single band with a minimum molecular mass of 63 kDa. Analytical gel-filtration, sedimentation-equilibrium and sedimentation-velocity experiments indicate that the purified enzyme exists in solution mainly as a dimer, containing 1 molecule non-covalently bound FAD/subunit. 3-Hydroxyphenylacetate 6-hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3-hydroxyphenylacetate is hydroxylated efficiently, yielding 2,5-dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4-dihydroxyphenylacetate and 4-hydroxyphenylacetate are partially hydroxylated, exclusively at the 6' (2') position. The physiological product 2,5-dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3-hydroxyphenylacetate 6-hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate. In the native state of enzyme, two sulfhydryl groups are accessible to 5,5'-dithiobis(2-nitrobenzoate). Titration with stoichiometric amounts of either 5,5'-dithiobis(2-nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3-hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol. The N-terminal amino acid sequence of the enzyme lacks the consensus sequence GXGXXG, found at the N-termini of all flavin-dependent external monooxygenases sequenced so far. The amino acid composition of 3-hydroxyphenylacetate 6-hydroxylase is also presented.
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PMID:Purification and characterisation of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacterium species. 193 54

Comparison of the amino acid sequences of several microsomal cytochrome P-450 reductases to the flavoprotein domain (BMR) of cytochrome P-450BM-3 has revealed that this class of flavoproteins contains evolutionarily conserved regions that are important for their interaction with nucleotide substrates and cofactors. In order to understand the properties of BMR, the region encoding this protein, beginning at residue Lys-472 of cytochrome P-450BM-3, was subcloned and expressed in Escherichia coli. The recombinant protein (more than 50% of host-soluble proteins) was purified to homogeneity using conventional purification procedures. BMR (Mr 66,000) showed typical flavoenzyme absorbance spectra, contained FAD and FMN in a stoichiometry of 1:1, and catalyzed reduction of several artificial electron acceptors with rates comparable to those of the microsomal NADPH-cytochrome P-450 oxidoreductase. Limited trypsinolysis of BMR, under non-denaturing conditions, revealed that the protease removed the NH2-terminal 122 residues. This region was postulated to contain amino acids that are important for FMN binding (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158). Consistent with this hypothesis, the major tryptic product of BMR (BMR-52, Mr 52,000) contained only FAD, in an equimolar ratio to the protein. Also, like the FMN-depleted microsomal NADPH-cytochrome P-450 oxidoreductase (Kurzban, G. P., Howarth, J., Palmer, G., and Strobel, H. W. (1990) J. Biol. Chem. 265, 12272-12279), BMR-52 was active for only catalyzing ferricyanide reduction. These data provide strong experimental evidence for a discrete multidomain structure of BMR, as proposed for the membrane-bound reductases, with an amino-terminal FMN binding region and carboxyl-terminal FAD- and NADPH binding regions. Thus, BMR strongly resembles the microsomal cytochrome P-450 reductase and offers an opportunity to better understand the structure-function relationships of this class of flavoproteins.
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PMID:Expression, purification, and properties of the flavoprotein domain of cytochrome P-450BM-3. Evidence for the importance of the amino-terminal region for FMN binding. 193 79

Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned.
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PMID:Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli. 197 42

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the dimeric flavoprotein glutathione reductase, Cys-47 (a cysteine residue forming an essential charge-transfer complex with enzyme-bound FAD) was converted to serine (C47S) and His-439 (required to facilitate protonation of the reduced glutathione) was converted to glutamine (H439Q). Both mutant genes were placed in the same plasmid, pHD, where each of them came under the control of a strong tac promoter. This was designed to achieve equal over-expression of both genes in the same E. coli cell. The parental homo-dimers show no (C47S) or very little (H439Q) activity as glutathione reductases. The formation in vivo of heterodimers, carrying one crippled and one fully functional active site, was detected by absorbance spectroscopy and fluorescence emission spectrometry of enzyme-bound FAD and by active site complementation. The fractional distribution of homo- and hetero-dimers was in accord with that expected for a random association of enzyme subunits. In a homo-dimer, the H439Q mutation leads to a big fall in the value of Km for NADPH which binds some 1.8 nm from the point of mutation (Berry, A., Scrutton, N.S. & Perham, R. N. Biochemistry 28, 1264-1269 (1989)). However, the one active site in the H439Q/C47S hetero-dimer exhibited kinetic parameters similar to those of the wild-type enzyme. Thus, the effect of the H439Q mutation must be retained within the active site that accommodates it and is not transmitted through the protein to the second active site across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo. 198 37

Mercuric reductase, a flavoprotein disulfide oxidoreductase, catalyzes the two-electron reduction of Hg(II) to Hg(0) by NADPH. As with all the members of this class of proteins, the enzyme is a dimer of identical subunits with two active sites per dimer, each composed of one FAD and catalytically essential residues from both subunits. In the enzyme from Tn501, these residues include, at a minimum, FAD and cysteines 135 and 140 from one subunit and cysteines 558' and 559' from the other. With this sort of active site arrangement, the enzyme seems perfectly set up for some type of subunit communication. In this report, we present results from several titrations, as well as kinetics studies, that, taken together, are consistent with the occurrence of subunit communication. In particular, the results indicate that pyridine nucleotide complexed dimers of the enzyme are asymmetric. Since the EH2-NADPH complex of the enzyme is the relevant reductant of Hg(II), these observations suggest that the enzyme may function asymmetrically during catalysis. An alternating sites model is proposed for the catalytic reduction of Hg(II), where both subunits of the dimer function in catalysis, but the steps are staggered and the subunits reverse roles after part of the reaction. An attractive feature of this proposal is that it provides a reasonable solution to the thermodynamic dilemma the enzyme faces in needing to both bind Hg(II) very tightly and reduce it.
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PMID:Communication between the active sites in dimeric mercuric ion reductase: an alternating sites hypothesis for catalysis. 200 50

The interaction between 2',5'-ADP and NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria was examined by titrating the enzyme with 2',5'-ADP, while the 31P-signals of 2',5'-ADP were being monitored by 31P-NMR. From the titration profile, the dissociation constant for the complex of the enzyme with 2',5'-ADP was estimated to be 0.22 +/- 0.05 mM. Adrenodoxin reductase was reconstituted with 13C-enriched FADs. The 13C-enriched FADs used were [2-13C]-, [4,10 alpha-13C2]-, and [4 alpha-13C]FAD. The 13C-NMR spectra of these reconstituted enzyme preparations showed 13C-resonance peaks corresponding to the enriched carbon atoms at 160.6 , 165.1, 136.6, and 152.4 ppm (2-, 4-, 4 alpha-, and 10 alpha-13C atoms, respectively). When 2',5'-ADP was bound to the reconstituted enzyme, these 13C-resonance peaks did not shift appreciably from those of the unbound enzyme, whereas in the complex of the reconstituted enzyme with NADP+, the signals for 4- and 10 alpha-13C shifted to higher fields by 2.1 and 0.7 ppm, respectively and the 4 alpha-13C signal shifted to a lower field by 1.4 ppm. These results suggest that in the complex of the enzyme with NADP+ the pyridine moiety is located in the vicinity of C(4 alpha)-C(4) region and that the pi-electron density of the 4 alpha-position of flavin is decreased in the enzyme-NADP+ complex. This argues in favor of the electron transfer from the dihydropyridine moiety of NADPH to the electron-deficient N(5) = C(4 alpha) region of flavin.
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PMID:On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P- and 13C-NMR. Use of 13C-enriched FAD as a probe. 201 63

FAD-dependent methaemoglobin reductases (MHR) were studied in red cells in heterozygous beta-thalassaemia to investigate how they related to low FAD-dependent glutathione reductase (GR). In contrast to GR, MHR activities were usually normal or increased. In particular, whether expressed in relation to haemoglobin or number of red cells, NADPH-MHR activity was markedly increased in most subjects, probably being a response to increased oxidative stress. Oral riboflavin had no effect on MHR activities, indicating saturation with FAD even though GR was deficient. A strong correlation between percent stimulation of GR by FAD and NADPH-MHR activity indicates that FAD is utilized by MHR at the expense of GR. This could be an important influence on GR in heterozygous beta-thalassaemia. Thus, the low activity resulting from an inherited deficiency of FAD is decreased further.
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PMID:Utilization of red-cell FAD by methaemoglobin reductases at the expense of glutathione reductase in heterozygous beta-thalassaemia. 204 24


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