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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

L-phenylalanine oxidase from Pseudomonas sp. P-501 was shown by isoelectric focusing and HPLC experiments to consist of two kinds of nonidentical subunits. The newly identified subunit is designated as alpha, and the larger subunit, which has been reported previously, as beta. The apparent molecular weight of alpha subunit was estimated to be 8200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the molecular mass of each subunits and their contents in the enzyme, the enzyme was shown to be composed to two alpha and two beta subunits. The amino acid sequence analysis of alpha subunit showed that the subunit consists of 92 amino acid residues and contains considerably hydrophobic arrangements and the candidate for the common sequence characteristic of the AMP binding in the FAD binding domain.
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PMID:New subunit in L-phenylalanine oxidase from Pseudomonas sp. P-501 and the primary structure. 810 68

Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH. 821 22

Mammalian electron-transferring flavoprotein (ETF) has been reported to consist of two non-identical subunits and one FAD. The present paper shows that ETF purified from pig kidney contains one more molecule, an AMP. ETF was denatured by guanidine hydrochloride and ultrafiltered for the purpose of removing proteins. The filtrate was analyzed by reverse-phase chromatography. Two peaks appeared on the chromatogram: they were identified as FAD and AMP, and their molar amounts were identical, indicating that ETF contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contain one AMP molecule. These results clearly demonstrate that ETF has an AMP-binding site in addition to the FAD-binding site. AMP-free apoETF was prepared by guanidine treatment of ETF. Mixing AMP-free apoETF, FAD, and AMP produced reconstituted ETF, which showed the same properties as native ETF. Mixing AMP-free apoETF and FAD produced AMP-free ETF, regardless of the coexistence of ATP or ADP: the AMP-binding site cannot bind FAD, ADP, or ATP. The enzymatic activity of the AMP-free ETF for electron transfer from substrate-reduced medium-chain acyl-CoA dehydrogenase to 2,6-dichlorophenolindophenol was identical to that of native ETF. This indicates that the AMP contained in holoETF has no apparent influence on this enzymatic activity. A role of AMP recognized in this study is that AMP facilitates the formation of holoETF from AMP-free apoETF, FAD, and AMP.
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PMID:Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site. 826 2

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) in the inner mitochondrial membrane accepts electrons from electron-transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64-kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF-QO and used these as probes to clone a full-length human ETF-QO cDNA. The deduced human ETF-QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin-like iron-sulfur clusters. A cleavable 33-amino-acid sequence is also predicted at the amino terminus of the 67-kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67-kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF-QO is synthesized as a 67-kDa precursor which is targeted to mitochondria and processed in a single step to a 64-kDa mature form located in the mitochondrial membrane. The detergent-solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron-sulfur cluster are properly inserted into the heterologously expressed protein.
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PMID:Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase. 830 95

Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 degrees C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.
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PMID:Discovery of a third coenzyme in sarcosine oxidase. 852 44

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric protein containing three different enzymes: noncovalent FAD, noncovalent NAD+, and covalently bound flavin which is released as 8 alpha-(N3-histidyl)riboflavin upon complete hydrolysis of the protein. The following results show that the covalent flavin is not at the FAD level, as previously proposed, but it is rather as 8 alpha-(N3- histidyl)FMN coenzyme. First, no AMP is released when the protein moiety is treated with phosphodiesterase or subjected to mild acid hydrolysis. The enzyme contains a total of 5 mol of phosphate. Only one phosphate is covalently bound. The other four phosphates are noncovalent and attributed to noncovalently bound FAD and NAD+. The 31P NMR spectrum of native enzyme exhibits resonances due to a single phosphate monoester an two pyrophosphates. Only a resonance due to phosphate monoester is observed after removal of the noncovalent cofactors and proteolytic digestion of the protein moiety. The 8 alpha-(N3-histidyl)FMN found in corynebacterial sarcosine oxidase represents a novel type of covalent flavin. Studies with sarcosine oxidases from Arthrobacter sp. and Pseudomonas sp. show that these heterotetrameric enzymes also contain covalently bound FMN plus noncovalently bound FAD and NAD+, similar to corynebacterial sarcosine oxidase. In contrast, two monomeric sarcosine oxidases (from Bacillus sp. and an unidentified microorganism) were found to contain only covalently bound FAD.
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PMID:Sarcosine oxidase contains a novel covalently bound FMN. 861 16

The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 micromol ferricyanide reduced x min(-1) x mg protein(-1) with xanthine as electron donor, which corresponds to approximately 30 micromol xanthine oxidized x min(-1) x mg protein(-1) with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)molybdopterin cytosine dinucleotide was identified, which indicates that molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S) clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.
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PMID:Characterization of xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica and identification of a molybdopterin-cytosine-dinucleotide-containing molybdenum cofactor. 870 91

A low molecular weight factor in a basal medium essential for erythrocytic Plasmodium falciparum development in a serum-free medium using a cell growth-promoting factor derived from adult bovine serum was detected. The factor was hypoxanthine. The optimal hypoxanthine concentration for parasite growth was between 15 and 120 microM. The contribution of hypoxanthine to increased parasite growth was clearly evident in cultures on day 4. Among various low molecular weight supplements tested, adenine, adenosine, AMP, ATP, cyclic AMP, guanine, guanosine, inosine, inosine monophosphate, xanthine, NAD, NADH, NADP, NADPH and deoxyguanosine triphosphate showed a similar effect to that of hypoxanthine in the serum-free culture system. On the other hand, the addition of uric acid, FAD, thymidine, uridine, orotic acid, deoxythymidine triphosphate, deoxycytidine triphosphate, deoxyadenosine triphosphate, ribose-1-phosphate, or ethanolamine was not beneficial to the parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements of the parasite.
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PMID:Hypoxanthine: a low molecular weight factor essential for growth of erythrocytic Plasmodium falciparum in a serum-free medium. 871 Apr 11

Abnormalities in calcium regulation, amyloid-beta-protein (A beta) production and oxidative metabolism have been implicated in Alzheimer's disease (AD). The use of cultured fibroblasts complement post-mortem and genetic approaches in clarifying the interaction of these processes and the underlying mechanism for the changes in AD. Definition of gene defects in particular Alzheimer families (FAD) permits elucidation of the role of those genetic abnormalities in altered signal transduction in cell lines from those families. Abnormalities in calcium regulation, ion channels, cyclic AMP, the phosphatidylinositide cascade and oxidative metabolism are well documented in fibroblasts from patients with primary genetic defects in the presenilins. Recent studies in AD fibroblasts that demonstrate abnormal secretion of A beta, a protein known to form the characteristic extracellular amyloid deposits in AD brain, further supports the use of these cells in AD research. Comparison of changes in calcium signaling, mitochondrial oxidation and A beta production in these cells suggests that changes in signal transduction including calcium may be a more consistent observation than altered A beta production in fibroblasts from some FAD families. An understanding of these abnormalities in fibroblasts may provide further insights into the pathophysiology of AD, new diagnostic measures and perhaps innovative therapeutic approaches.
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PMID:Altered oxidation and signal transduction systems in fibroblasts from Alzheimer patients. 876 36

Electron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (alpha and beta, molecular weights of 33,000 and 29,000) and two small molecules, FAD and AMP. In this study, in vitro refolding and unfolding of the subunits of ETF were carried out with urea as the denaturing reagent. The refolding reaction of alpha and beta was revealed to proceed kinetically in two steps: D in equilibrium with I-->N, where D,I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of alpha and beta, described below, are consistent with the concept of the so-called "molten globule state," which is frequently observed in protein refolding. (i) The conversion between D and I was very rapid. (ii) The I form showed as much secondary structure as the N form as judged from the far-UV circular dichroism. (iii) The solvent accessibility of the I form, estimated by the analysis of equilibrium unfolding experiments, was intermediate between those of the D and N forms. (iv) The standard free energy of the I form is almost the same as that of the D form. The refolding reaction progressed more slowly and the environment of the tryptophan chromophore was changed more drastically in beta refolding that in alpha refolding. We previously reported that the reconstitution of holoETF from denatured subunits is speeded up by increasing the AMP concentration. In this study, the effects of AMP, FAD, and the other subunit on the single subunit folding were examined, but no effect was detected. This result suggests that AMP plays a role in a later process, namely, assembly of the four components (refolded alpha and beta, FAD, and AMP).
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PMID:In vitro refolding and unfolding of subunits of electron-transferring flavoprotein: characterization of the folding intermediates and the effects of FAD and AMP on the folding reaction. 888 11


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