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PubMed2003

A conserved domain in prokaryotic bifunctional FAD synthetases can potentially catalyze nucleotide transfer.

Biosynthesis of flavin adenine dinucleotides in most prokaryotes is catalyzed by a family of bifunctional flavin adenine dinucleotide (FAD) synthetases. These enzymes carry out the dual functions of phosphorylation of flavin mononucleotide (FMN) and its subsequent adenylylation to generate FAD. Using various sequence analysis methods, a new domain has been identified in the N-terminal region that is well conserved in all the bacterial FAD synthetases. We also identify remote similarity of this domain to the nucleotidyl transferases and, hence, this domain is suggested to be invloved in the adenylylation reaction of FAD synthetases.

Amino Acid Sequence↗
PubMed2003

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode.

The electrochemical properties (such as the values of the formal potentials, the dependence of the formal potentials on solution pH, the reversibility of the electrochemical process) of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) adsorbed on a titanium electrode were dependent on the electrolyte. The formal potentials of adsorbed FMN and FAD in phosphate, HEPES and PIPES buffers at pH 7 were similar to those for dissolved flavins (-460 to -480 mV vs. SCE) and changed linearly with a slope of about 52 mV per pH unit in the pH region 3 to 8. In TRIS buffer, the formal potentials of adsorbed FMN and FAD were also pH-dependent, however, with invariance in the pH range 4.5 to 5.5. In non-buffered solutions (KCl, LiCl, NaCl, CsCl, CaCl(2), Na(2)SO(4) at different concentrations), the electrochemical behavior of adsorbed FMN and FAD differed from that of dissolved flavins and was dependent on the electrolyte (especially at pH 4.5 and pH 5). Under certain conditions (electrolyte, concentration, pH), a two-step oxidation of FMN could be observed.

Buffers↗
PubMed1988

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase.

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1989

The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation.

p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2003

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins.

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.

Animals↗
PubMed2004

Use of 8-substituted-FAD analogues to investigate the hydroxylation mechanism of the flavoprotein 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) is a flavoprotein that catalyzes the oxygenation of MHPC to form alpha-(N-acetylaminomethylene)-succinic acid. Although formally similar to the oxygenation reactions catalyzed by phenol hydroxylases, MHPCO catalyzes the oxygenation of a pyridyl derivative rather than a simple phenol. Therefore, in this study, the mechanism of the reaction was investigated by replacing the natural cofactor FAD with FAD analogues having various substituents (-Cl, -CN, -NH(2), -OCH(3)) at the C8-position of the isoalloxazine. Thermodynamic and catalytic properties of the reconstituted enzyme were investigated and found to be similar to those of the native enzyme, validating that these FAD analogues are reasonable to be used as mechanistic probes. Dissociation constants for the binding of MHPC or the substrate analogue 5-hydroxynicotinate (5HN) to the reconstituted enzymes indicate that the reconstituted enzymes bind well with ligands. Redox potential values of the reconstituted enzymes were measured and found to be more positive than the values of free FAD analogues, which correlated well with the electronic effects of the 8-substituents. Studies of the reductive half-reaction of MHPCO have shown that the rates of flavin reduction by NADH could be described as a parabolic relationship with the redox potential values of the reconstituted enzymes, which is consistent with the Marcus electron transfer theory. Studies of the oxidative half-reaction of MHPCO revealed that the rate of hydroxylation depended upon the different analogues employed. The rate constants for the hydroxylation step correlated with the calculated pK(a) values of the 8-substituted C(4a)-hydroxyflavin intermediates, which are the leaving groups in the oxygen transfer step. It was observed that the rates of hydroxylation were greater when the pK(a) values of C(4a)-hydroxyflavins were lower. Although these results are not as dramatic as those from analogous studies with parahydroxybenzoate hydroxylase (Ortiz-Maldonado et al., (1999) Biochemistry 38, 8124-8137), they are consistent with the model that the oxygenation reaction of MHPCO occurs via an electrophilic aromatic substitution mechanism analogous to the mechanisms for parahydroxybenzoate and phenol hydroxylases.

Apoenzymes↗
PubMed2004

Structural intermediate in the photocycle of a BLUF (sensor of blue light using FAD) protein Slr1694 in a Cyanobacterium Synechocystis sp. PCC6803.

Slr1694 in Synechocystis sp. PCC6803 is a family of blue-light photoreceptors based on flavin adenine dinucleotide (FAD) called BLUF (sensor of blue light using FAD) proteins, which include AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis. Illumination of dark-state Slr1694 at 15 degrees C reversibly induced a signaling light state characterized by the red shift in the UV-visible spectrum and by the light-induced Fourier transform infrared (FTIR) difference spectrum for structural changes of a bound flavin and apo protein. Illumination at the medium-low temperature (-35 degrees C) led to the red shift in the UV-visible spectrum despite some small difference in the light-induced changes. In contrast, the -35 degrees C illumination resulted in a completely different light-induced FTIR spectrum, in which almost all of the bands were suppressed with the exception of the bands for the change of C4=O bonding of the FAD isoalloxazine ring. The C4=O bands were induced at -35 degrees C with almost the same intensity, but the band frequency for the light state was upshifted by 6 cm(-)(1). The changes in frequency of the light-state C4=O band and in amplitude of other bands showed the same temperature dependence with a half-change temperature at approximately -20 degrees C. It was indicated that the light-induced structural changes of apo protein and FAD were inhibited at low temperature with the exception of the change in hydrogen bonding to the C4=O group. The light-induced formation of the FTIR bands was similarly inhibited by sample dehydration. We discussed the possibility that this constrained light state is a trapped intermediate state in the photocycle of Slr1694.

Bacterial Proteins↗
PubMed1996

Role of Ser457 of NADPH-cytochrome P450 oxidoreductase in catalysis and control of FAD oxidation-reduction potential.

Site-directed mutagenesis of Ser457 of NADPH-cytochrome P450 oxidoreductase demonstrates that this residue plays a major role in both hydride transfer from NADPH to FAD and modulation of FAD redox potential. Substitution of Ser457 with alanine or cysteine decreases the rates of reduction of the substrates cytochrome c and potassium ferricyanide approximately 100-fold, while substitution with threonine produces a 20-fold decrease in activity. No changes are observed in k(m)NADPH, KiNADP+, or flavin content, indicating that these substitutions have no effect on cofactor binding but affect catalysis only. k(m)cyt c values are decreased in parallel with the observed decreases in the rates of the reductive half-reaction. Stopped-flow studies with the S457A mutant show a 100-fold decrease in the rate of flavin reduction. The primary deuterium isotope effect on Kcat for cytochrome c reduction increases from 2.7 for the wild-type enzyme to 9.0 for the S457A mutant, consistent with a change in the rate-determining step from NADP+ release in the wild-type enzyme to hydride transfer in the S457A mutant. The primary deuterium isotope effect on K1 for flavin reduction at high ionic strength (I = 535 mM) increases from 12.2 for the wild-type enzyme to > 20 for the S457A mutant, consistent again with an increase in the relative rate limitation of hydride transfer. Furthermore, anaerobic titration of S457A indicates that the redox potential of the FAD semiquinone has been decreased. Data presented in this study support the hypothesis that Ser457 is involved in hydrogen bonding interactions which stabilize both the transition state for hydride transfer and the reduced FAD.

Base Sequence↗
PubMed1997

Hyperaccumulation of FAD-linked presenilin 1 variants in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.

Actins↗
PubMed2002

A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p.

Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum. The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases. Two pairs of cysteine residues are required for Erv2p activity. The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD. The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.

Amino Acid Motifs↗
PubMed1998

Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD.

Anti-mitochondrial antibodies (anti-M7) in sera from patients with dilated cardiomyopathy and myocarditis recognize, besides mitochondrial antigens, bacterial sarcosine dehydrogenase. The common target antigen was identified as the covalently bound FAD of mitochondrial and bacterial flavoenzymes. Thus, anti-M7-positive serum reacted on Western blots exclusively with covalently flavinylated enzymes. The antigenic specificity of anti-M7 sera was reproduced by an antiserum raised in rabbits with 6-hydroxy-D-nicotine oxidase. The heart mitochondrial membrane antigen recognized by anti-M7 serum was identified as the flavoprotein subunit of succinate dehydrogenase, the antigens in rat liver mitochondrial matrix as the flavoenzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase. Anti-M7 serum contained a specific anti-flavoenzyme antibody fraction. Nanomolar concentrations of FAD and riboflavin inhibited the immune reaction on Western blots and in ELISA, and incubation with FAD-agarose depleted the anti-M7 activity of the serum. N-terminally deleted dimethylglycine dehydrogenase proteins were only immunoprecipitated by anti-M7 sera when the FAD was covalently incorporated. An affinity constant (KD) of 10(-8) M was established for the anti-flavoenzyme antibodies by competitive ELISA. Of patients with cardiomyopathy and myocarditis, 36% and 25%, respectively, were anti-flavoenzyme-positive by Western blot and ELISA, but only two of 15 patients with other heart diseases and none of 50 healthy controls.

Animals↗
PubMed1997

Human presenilin-1, but not familial Alzheimer's disease (FAD) mutants, facilitate Caenorhabditis elegans Notch signalling independently of proteolytic processing.

The majority of cases with familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes show considerable sequence similarity to the sel-12 gene of Caenorhabditis elegans, which has been postulated to function in the facilitated signalling by lin-12 and glp-1. In order to analyse the functional conservation of the presenilins, we introduced the human PS-1 cDNA, as well as clinical and deletion mutant proteins, into sel-12 mutant animals and tested their potential to rescue the egg-laying defect. Human PS-1 expressed from the sel-12 promoter fully rescued the sel-12 phenotype, whereas two missense mutations, C410Y and A246E, identified in pedigrees with FAD, exhibited a strongly decreased rescuing activity. The large hydrophilic loop and transmembrane domain 7 are required for the biological activity of PS-1. PS-1 protein was proteolytically cleaved in C. elegans as it is in human cells. A PS-1 splice variant (FAD mutation deltaexon9) that does not undergo proteolytic cleavage also substituted for sel-12. The conservation of function of human PS-1 and C. elegans sel-12 suggests that presenilin proteins are required, directly or indirectly, for the proper operation of the Notch signalling pathway. FAD-associated mutant proteins tested showed different rescuing activities, indicating that they might affect different functional or regulatory aspects of PS-1. Proteolytic processing is not a prerequisite for PS-1 function in C. elegans.

Alzheimer Disease↗
PubMed1991

Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.

Amino Acid Oxidoreductases↗
PubMed2001

alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone.

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.

Arginine↗
PubMed2001

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family.

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

Amino Acid Motifs↗
PubMed2003

Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD.

A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 degrees C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group.

Amino Acid Sequence↗
PubMed2003

Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription.

Protein enzymes frequently recruit small molecule coenzymes to perform a variety of biochemical reactions. While the catalytic activities of RNA have been expanding rapidly, a similar strategy for RNA to utilize coenzymes and to increase its functional capabilities has yet to be demonstrated. A general in vitro transcription procedure has been developed to efficiently prepare RNA with coenzymes CoA, NAD and FAD covalently attached to the 5' end. These adenosine-containing coenzymes initiate transcription under the T7 class II promoter by T7 RNA polymerase. In addition to the three coenzymes, other adenosine-containing molecules may be incorporated into the first nucleotide position of RNA as well. This method provides easy access to CoA-, NAD- and FAD-RNA, which may find broad applications in generating coenzyme- utilizing ribozymes. In addition, both oxidized FAD and reduced NADH are highly fluorescent. NADH-RNA and FAD-RNA can therefore be used as probes for DNA/RNA detection and for structural investigation of RNA function by fluorescence spectroscopy.

Adenosine↗
PubMed1981

Glutathione reductase from human erythrocytes: amino-acid sequence of the structurally known FAD-binding domain.

Glutathione reductase (Mr 2 x 52 500), a flavoenzyme of known three-dimensional structure, catalyses the reduction of glutathione disulfide by NADPH. This paper describes the primary structure of the FAD-binding domain which ranges from AcAla-1 to Gly-157. The three CNBr-produced fragments (69, 10 and 80 residues) of the domain were fractionated further by enzymatic and chemical methods; isolated peptides were sequenced mainly by automatic solid-phase Edman degradation. The tryptic peptides were overlapped by chymotryptic peptides. A fragment which results from cleavage at the acid-labile bond between Asp-135 and Pro-136 supplied peptides for overlapping the CNBr-produced fragments. In addition, many peptides were ordered and overlapped by computerized comparison with a complete sequence guessed from the electron density map. With one exception the computer method and the chemical alignment gave the same results. The sequence data are discussed in the light of the secondary and tertiary structure (Schulz et al. (1978) Nature (Lond.) 273, 120--124]. The 17 N-terminal residues are not visible in the electron density map. Consequently our numbering scheme differs from that of Schulz et al. by approximately 20 residues. Acetylation of the N terminus and an unusual composition of the following residues may serve to protect the loose N-terminal section of the protein against proteolysis in situ. The four cysteinyl residues of the FAD domain are of special interest. Cys-2 at the tip of the N-terminal extension is likely to be involved in the aggregation behaviour of glutathione reductase. Cys-58 and Cys-63 (formerly Cys-41 and Cys-46) represent the enzyme's redox-active dithiol. Cys-90 with its location at the twofold axis forms a disulfide bridge with Cys-90 of the other peptide chain of the enzyme. This might be related to the fact that both peptide chains contribute to each of the two active centers. In view of the interchain disulfide bridge glutathione reductase should be regarded as a monomeric protein. The sequence of the FAD-binding domain was compared with the sequence of the NADPH-binding domain of glutathione reductase using a computer program. As discussed, the scarcity of sequence similarities does not argue against the assumption that the two nucleotide-binding domains of glutathione reductase originated by gene duplication. The pyrophosphate moiety of FAD binds to a part of the polypeptide chain which in geometric structure, in topology and in sequence resembles the phosphate loops of other nucleotide-binding proteins and of flavodoxin. Using the phosphate loop as a reference, the N-terminal sequence of five flavoproteins can be aligned. The results of Williams et al. on the sequence of lipoamide dehydrogenase (EC 1.6.4.3) and our data on glutathione reductase (EC 1.6.4.2) show clearly that these two mechanistically similar enzymes possess homologous structures.

Amino Acid Sequence↗