FAD Prosthetic Group Research Articles

The Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus--redox states of the FAD prosthetic group and mechanism of Ag+ inhibition.

The FAD prosthetic group of the Na+-motive NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio alginolyticus was investigated by ultraviolet-visible and fluorescence spectroscopy. The reduction of Na+-NQR by excess NADH in the presence of 6-13 microM O2 resulted in the formation of the blue flavosemiquinone radical. If the concentration of dioxygen was further reduced to 0.1 microM O2, neither the reduction of Na+-NQR by NADH nor its reoxidation with ubiquinone-1 (Q-1) yielded a stable flavosemiquinone in equilibrium with reductant or oxidant, respectively, but the fully reduced (Fl(red)H2) or oxidized flavin (Fl(ox)) prevailed. During reoxidation of Fl(red)H2 with Q-1, the intermediate formation of an absorbance band around 800 nm was observed, which was tentatively assigned as the Fl(red)H(-)-NAD+ charge-transfer complex. Complete reoxidation of Fl(red)H2 in Na+-NQR was achieved by a fivefold excess of Q-1 over NADH. These results indicated that only a small fraction of FAD was in the flavosemiquinone redox state during turnover to mediate the electron transfer between the hydride donor, NADH, and the one-electron acceptor [2Fe-2S]. The titration of Na+-NQR with Ag+, a specific inhibitor, was followed by the fluorescence emission spectra of FAD (Fl(ox)). The addition of Ag+ resulted in a marked increase of the flavin fluorescence (16% at 200 nM Ag+), with half-maximal saturation at approximately 50 nM Ag+, indicating dissociation of FAD from the enzyme. The increase in fluorescence intensity correlated with the loss of enzyme activity. Gel filtration of the Ag+-treated Na+-NQR confirmed that FAD had been displaced from the holo-enzyme.

Catalytic Properties of NAD(P)H:Quinone Oxidoreductase-2 (NQO2), a Dihydronicotinamide Riboside Dependent Oxidoreductase

Human NAD(P)H:quinone acceptor oxidoreductase-2 (NQO2) has been prepared using anEscherichia coliexpression method. NQO2 is thought to be an isoform of DT-diaphorase (EC 1.6.99.2) [also referred to as NAD(P)H:quinone acceptor oxidoreductase] because there is a 49% identity between their amino acid sequences. The present investigation has revealed that like DT-diaphorase, NQO2 is a dimer enzyme with one FAD prosthetic group per subunit. Interestingly, NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. It catalyzes a two-electron reduction of quinones and oxidation–reduction dyes. One-electron acceptors, such as potassium ferricyanide, cannot be reduced by NQO2. This enzyme also catalyzes a four-electron reduction, using methyl red as the electron acceptor. The NRH-methyl red reductase activity of NQO2 is 11 times the NADH-methyl red reductase activity of DT-diaphorase. In addition, through a four-electron reduction reaction, NQO2 can catalyze nitroreduction of cytotoxic compound CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. NQO2 is 3000 times more effective than DT-diaphorase in the reduction of CB 1954. Therefore, NQO2 is a NRH-dependent oxidoreductase which catalyzes two- and four-electron reduction reactions. NQO2 is resistant to typical inhibitors of DT-diaphorase, such as dicumarol, Cibacron blue, and phenindone. Flavones are inhibitors of NQO2. However, structural requirements of flavones for the inhibition of NQO2 are different from those for DT-diaphorase. The most potent flavone inhibitor tested so far is quercetin (3,5,7,3′,4′-.6pentahydroxyflavone). It has been found that quercetin is a competitive inhibitor with respect to NRH (Ki= 21 nM). NQO2 is 43 amino acids shorter than DT-diaphorase, and it has been suggested that the carboxyl terminus of DT-diaphorase plays a role in substrate binding (S. Chenet al., Protein Sci.3, 51–57, 1994). In order to understand better the basis of catalytic differences between NQO2 and DT-diaphorase, a human NQO2 with 43 amino acids from the carboxyl terminus of human DT-diaphorase (i.e., hNQO2–hDT43) has been prepared. hNQO2–hDT43 still uses NRH as an electron donor. In addition, the chimeric enzyme is inhibited by quercetin but not dicumarol. These results suggest that additional region(s) in these enzymes is involved in differentiating NRH from NAD(P)H.

Direct Electrochemistry of the Flavin Domain of Assimilatory Nitrate Reductase: Effects of NAD+and NAD+Analogs

Direct electrochemical studies, utilizing two voltammetric methods—square-wave voltammetry (SWV) and cyclic voltammetry (CV)—have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+analogs. The reduction potentials (E°′) of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25°C), the two-electron reduction potential for the FAD/FADH2couple has been determined to be −274 and −257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be −234 and −227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+analogs have been determined to be as follows: NAD+(E°′ = −192 mV), 5′-ADP ribose (E°′ = −199 mV), ADP (E°′ = −154 mV), AMP (E°′ = −196 mV), adenosine (E°′ = −192 mV), adenine (E°′ = −220 mV), and NMN (E°′ = −208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E°′ = −268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.

Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis

The protein p64k from the surface of the Neisseria meningitidis bacteria has been characterized as a two-domain protein. It contains a dihydrolipoamide dehydrogenase domain of 482 residues, involving a FAD prosthetic group as a cofactor, and a smaller lipoic acid binding domain of 86 residues. The two domains are joined by a flexible segment rich in alanine and proline residues. The structure of the dihydrolipoamide dehydrogenase domain was determined by X-ray diffraction. It was solved by a combination of molecular replacement and multiple isomorphous replacement techniques and refined to 2.7 Å resolution. In the crystal, the recombinant p64k mimics the functional homo-dimer by using one of the crystallographic 2-fold axes. The reactive disulphide bridge Cys161-Cys166 is in the oxidised state and the FAD is bound in an extended conformation. This main domain contains the major antigenic determinant of the protein, an extended loop of 32 residues at the surface of the protein. A mis-attribution at residue 553 in the sequence has been detected by inspection of electron density maps and the geometry. However, when compared to the other dihydrolipoamide dehydrogenases, there are some significant differences: (1) an unusual number of cis-proline residues and (2) a new motif built around a 2-fold axis by the sulphur atoms of residues Met558, Cys560 and their symmetry related equivalents.

AlphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein.

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.

Solubilization and Characterization of a Thyroid Ca2+‐Dependent and NADPH‐Dependent K3Fe(CN)6 Reductase

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.

The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 A resolution.

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved trypanocidal drugs. We present details of the structure of TR from the human pathogen Trypanosoma cruzi, the agent responsible for Chagas' disease or South American trypanosomiasis. The structure has been solved by molecular replacement, using as the starting model the structure of the enzyme from the nonpathogenic Crithidia fasciculata, and refined to an R-factor of 18.9% for 53,868 reflections with F > or = sigma F between 8.0 and 2.3 A resolution. The model comprises two subunits (968 residues), two FAD prosthetic groups, two maleate ions, and 419 water molecules. The accuracy and geometry of the enzyme model is improved with respect to the C. fasciculata enzyme model. The new structure is described and specific features of the enzyme involved in substrate interactions are compared with previous models of TR and related glutathione reductases from human and Escherichia coli. Structural differences at the edge of the active sites suggest an explanation for the differing specificities toward glutathionylspermidine disulfide.

The Flavin Reductase Activity of the Flavoprotein Component of Sulfite Reductase from Escherichia coli: A NEW MODEL FOR THE PROTEIN STRUCTURE

Sulfite reductase (SiR) from Escherichia coli has a alpha 8 beta 4 subunit structure, where alpha 8 is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups. It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates. The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction. A plasmid containing cysJ gene, coding for the alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha is the site of free flavin reduction. The overproducer allows a fast and simple preparation of large amounts of the flavoprotein. Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism. NADP+ inhibition studies show that both substrates, NADPH and free flavins, bind to the same site. While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity. In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per alpha subunit. This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per alpha subunit.

Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

The effect of N-bromosuccinimide on ferredoxin: NADP + oxidoreductase

Treatment of spinach leaf ferredoxin:NADP + oxidoreductase (FNR) with N-bromosuccinimide (NBS), under conditions where approximately one tryptophan residue per enzyme was modified, resulted in a loss of between 80 and 85% of the activity of the enzyme when electron transfer from NADPH to either ferredoxin or 2,6-dichlorophenol-indophenol was measured. Amino acid analysis revealed no detectable modification by NBS of any FNR amino acids other than tryptophan. Complex formation with ferredoxin, but not with NADP +, prevented both the inhibition of activity and the modification of tryptophan caused by the treatment with NBS. Modification of one FNR tryptophan residue had no significant effect on the K m values of the enzyme for either ferredoxin or NADPH or on the binding constants for the FNR complexes with either ferredoxin or NADP +. NBS treatment had only very small effects on the absorbance and circular dichroism spectra of FNR and did not significantly affect either the oxidation-reduction midpoint potential of the FAD prosthetic group of the enzyme or inhibit the reduction of the FAD group by NADPH. These results raise the possibility that a tryptophan residue may play a role in the electron transfer between the FAD of FNR and the enzyme substrate, ferredoxin.

Trypanothione reductase from Leishmania donovani. Purification, characterisation and inhibition by trivalent antimonials.

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 microM and 9 microM, respectively, the former yielding a kcat/Km of 5.0 x 10(6) M-1 s-1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.

Trypanothione Reductase from Leishmania donovani. Purification, Characterisation and Inhibition by Trivalent Antimonials

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 μM and 9 μM, respectively, the former yielding a kcat/Km of 5.0×106 M−1 s−1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.

Crystal Structure of NADH-Cytochrome b5 Reductase from Pig Liver at 2.4 .ANG. Resolution

The three-dimensional structure of NADH-cytochrome b5 reductase from pig liver microsomes has been determined at 2.4 A resolution by X-ray crystallography. The molecular structure reveals two domains, the FAD binding domain and the NADH domain. A large cleft lies between these two domains and contains the binding site for the FAD prosthetic group. The backbone structure of the FAD binding domain has a great similarity to that of ferredoxin-NADP+ reductase [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60-65], in spite of the relatively low sequence homology (about 15%) between the two enzymes. On the other hand, the structure of the NADH domain has several structural differences from that of the NADP+ domain of ferredoxin-NADP+ reductase. The size of the cleft between the two domains is larger in NADH-cytochrome b5 reductase than in ferredoxin-NADP+ reductase, which may be responsible for the observed difference in the nucleotide accessibility in the two enzymes.

S-2-Bromo-Acyl-CoA Analogs Are Affinity Labels for the Medium-Chain Acyl-CoA Dehydrogenase from Pig-Kidney

S-2-Br-hexanoyl-CoA and the branched chain isomer S-2-Br-4-methyl-pentanoyl-CoA are affinity labels of the medium-chain acyl-CoA dehydrogenase from pig kidney. The straight chain thioester is both a substrate and an irreversible inhibitor of the dehydrogenase. Inactivation of the enzyme is biphasic and is half-complete in 4 min at pH 6.5, 25°C. Although S-2-Br-hexanoyl-CoA can partially reduce the FAD prosthetic group of the dehydrogenase, inactivation results from attachment of one molecule of inhibitor per subunit of the oxidized enzyme. The branched chain analogue is a very weak substrate of the dehydrogenase (0.1% that of octanoyl-CoA), but is almost as effective an inhibitor of the dehydrogenase. Incubation experiments with [14C]S-2-Br-methyl-pentanoyl-CoA followed by the isolation of radiolabeled peptide show that modification of the active site base, GLU376, is responsible for enzyme inactivation. The data are compatible with a simple nucleophilic attack of the carboxylate base on the C-2 atom of these 2-Br-analogues.

Polyamine oxidase activity and growth in human cancer cells

lnterconversion of the polyamines, spermine + spermidine + putrescine, requires the N 1 -spermidine/spermine acetyltransferase (N1-SAT) and polyamine oxidase (PA01 enzymes. This process is used, in conjunction with biosynthesis and transport, to maintain the polyamine balance of the cell. N1-SAT is the rate-limiting step in the pathway and provides N1 -acetylpolyamine derivatives, the prefered substrate for PA0 [ll. PA0 contains a tightly bound FAD prosthetic group and acts on the secondary amine group of spermidine and spermine yielding spermidine and putrescine, respectively, along with 3-acetamidopropanal and hydrogen peroxide 121. Polyamine concentrations in cancerous tissue are high in comparison to normal tissue of the same type. High polyamine concentrations are also associated with rapidly dividing cells, suggesting alterations in either polyamine transport and/or polyamine metabolism are strongly associated with cell growth 131. As well as being substrates for PA0 acetylpolyamines have been suggested to be involved in excretion of polyamines 141. In this study we examined PA0 activity in a number of human tumour cell types and its relationship to cell growth. T47-D (breast carcinoma), G402 (renal leiomyoblastoma), and H T l l 5 (colonic carcinoma) cells were grown as monolayer cultures at a seeding density of 2 .5~104 celllcm2 (T47-D and G402) and 2.8~104 cell/cm2 (HT115). MOLT4-B (T leukaemia) cells were grown in suspension at 3x1 05 cells/ml. Culture medium was DMEM supplemented with 10% HS (HT1151, 10% FCS (G4021, 5% FCS & 1 % insulin (T47-D), or in RPMl with 10% FCS (MOLT4-B). Medium was changed every 48h where required. Cells were harvested at the appropriate growth condition for each cell type and the cells homogenised in Tris-HCI pH7.4 + 0.1 % Triton-X-l OO. PA0 activity was determined by a modification of the method of Table 1. PA0 activity in human tumour cells

An optical biosensor based upon glucose oxidase immobilized in sol-gel silicate matrix.

Glucose oxidase immobilized in a transparent silicate gel prepared by a sol-gel method was used as a simple solid-state optical biosensor for glucose. The sensor was based upon the measurement of initial rates of reduction of the FAD prosthetic group of the enzyme in the presence of various concentrations of glucose. The analytical range of the sensor was 1-100 mM, and the measurement time was 2 min. The enzyme was considerably protected by the silicate matrix against leaching, thermal inactivation and even H2O2-dependent inactivation. The sensor was stable in daily use for 6 months.

Construction and expression of a flavocytochrome b5 chimera.

A gene has been constructed coding for a chimeric flavocytochrome b5 protein that comprises the soluble domain of rat hepatic cytochrome b5 as the NH2-terminal portion of the chimera and the flavin-containing domain of spinach assimilatory NADH:nitrate reductase as the C terminus. The chimeric protein has been expressed in Escherichia coli and purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose, anion-exchange chromatography, and fast protein liquid chromatography gel filtration with an estimated molecular mass of 43 kDa from polyacrylamide gel electrophoresis. Visible and fluorescence spectroscopy indicated the purified protein contained both a b-type cytochrome and FAD prosthetic groups. The chimeric hemoflavoprotein immunologically cross-reacted with both anti-rat cytochrome b5 and anti-spinach nitrate reductase polyclonal antibodies, indicating the conservation of antigenic determinants from both native domains. NH2-terminal and internal amino acid sequencing of the native and CNBr-digested protein confirmed the presence of peptides derived from both the heme- and flavin-binding portions of the sequence which were identical to the deduced amino acid sequence. The chimera exhibited both NADH: ferricyanide reductase and NADH:cytochrome c reductase activities with Vmax values of 88 and 37 mumol of NADH consumed per min/nmol of heme (mu = 0.05 and pH 7.0) and Km values of 2.1, 32, and 1.4 microM for NADH, ferricyanide, and cytochrome c, respectively. This work represents the first successful bacterial expression of a mammalian-plant chimeric metalloflavoprotein. The chimera exhibited properties extremely similar to those of the native cytochrome b5 heme and spinach nitrate reductase FAD components.

Structure of trypanothione reductase from Crithidia fasciculata at 2.6 A resolution; enzyme-NADP interactions at 2.8 A resolution.

Trypanothione reductase is an FAD-dependent disulfide oxidoreductase which catalyses the reduction of trypanothione using NADPH as co-factor. The enzyme is unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved antitrypanocidal drugs. We present details of the structure of trypanothione reductase from Crithidia fasciculata solved by molecular replacement, using human glutathione reductase as a search model, and refined to an R factor of 16.1% with data between 8.0 and 2.6 A resolution. The model comprises two subunits (one containing 487 residues, the other 486), an FAD prosthetic group, plus 392 solvent molecules. The last four C-terminal residues are not seen in either subunit and the density is poor for the N-terminal residue of subunit B. The model has a root-mean-square deviation from ideality of 0.016 A for bond lengths and 3.2 degrees for bond angles. Each subunit was independently refined in the latter stages of the analysis but the subunits remain similar as indicated by the root-mean-square deviation of 0.35 A for C(alpha) atoms. Trypanothione reductase has 36% sequence identity with human glutathione reductase and the root-mean-square deviation between the 462 C(alpha) atoms in the secondary structural units common to the two proteins is 1.1 A. However, there are large differences in the loop regions and significant shifts in the orientation of the four domains within each subunit. Domain II, which binds the dinucleotide co-factor, and domain IV, which forms the interface between the two subunits, are both rotated by approximately 5 degrees with respect to domain I, which binds the FAD moiety, when compared with glutathione reductase. Crystals of trypanothione reductase have been soaked in the dinucleotide co-factor NADPH and N(1)-glutathionylspermidine disulfide substrate and the structure of the resulting complex determined at 2.8 A resolution. Strong density is observed for the adenosine end of the co-factor which forms many charged interactions with the protein though the density for the nicotinamide moiety is more diffuse. The mode of binding indicates that NADP is bound to the enzyme in a similar conformation to that observed with human glutathione reductase.

A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase.

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.

Arabidopsis thaliana NAPHP Thioredoxin Reductase : cDNA Characterization and Expression of the Recombinant Protein in Escherichia coli

Using a clone characterized in the course of a random sequencing programme of Arabidopsis thaliana, two cDNAs encoding plant type cytosolic NADPH-dependent thioredoxin reductase (NTR) have been isolated. Their sequence homology with Escherichia coli NRT (the only thioredoxin reductase of known primary structure) is about 45%. In addition, analysis of the sequence of the encoded polypeptide (333 amino acids) reveals that several motifs are conserved in the FAD, central and NADPH binding domains, suggesting a similar folding of the protein. Definitive proof that the clone ATTHIREDB indeed encodes NTR was obtained by expressing the recombinant protein in E. coli cells. It was observed that plant type NTR was strongly overproduced (about 10 mg homogeneous protein could be purified per liter of culture). The recombinant enzyme is homodimeric, each subunit containing an FAD prosthetic group. Recombinant plant type NTR is as effective as E. coli NTR in the DTNB (5,5′-dithiobis nitrobenzoic acid) reduction reaction, but its affinity for thioredoxin substrates was strikingly different. These results are discussed in relation to the primary structures of NADPH thioredoxin reductases.