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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent (Km = 8 microM) reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called "glutathione-induced thiol-reductase activity' previously observed in G. duodenalis.
Mol Biochem Parasitol 1996 Dec 20
PMID:A thioredoxin reductase-class of disulphide reductase in the protozoan parasite Giardia duodenalis. 902 54

Protoporphyrinogen oxidase (E.C.1.3.3.4) (PPO) catalyzes the penultimate step in the heme biosynthetic pathway. Deficiency in activity of this enzyme results in the human genetic disease variegate porphyria. Herein we detail the cloning, expression, purification and characterization of the normal and variegate porphyria forms of human PPO. The cDNA sequence for human ppo is approximately 1.8 kb in length and codes for a protein of 477 amino acids. This protein, which does not contain a typical cleavable mitochondrial targeting sequence, is approximately 51 kDa and contains a putative dinucleotide binding motif near the amino terminus. The active enzyme is a homodimer and contains an FAD. Attachment of a six his amino terminal tag allows for the rapid and efficient purification of approximately 10 mg of enzyme from one liter of E. coli culture. Three variegate porphyria mutant PPO enzymes were expressed and characterized. These mutations, R59W, R168C and A433P, result in decreased enzyme activity by causing a decrease in kcat without a significant change in Km for the substrate protoporphyrinogen IX. Purified R59W lacks the FAD cofactor which may be explained by the fact that this mutation resides within the dinucleotide binding motif of PPO.
Cell Mol Biol (Noisy-le-grand) 1997 Feb
PMID:Characteristics of human protoporphyrinogen oxidase in controls and variegate porphyrias. 907 90

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.'.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa, exhibiting absorption and fluorescence spectra typical for a flavoprotein and similar to the SQR from the cyanobacterium Oscillatoria limnetica. From N-terminal and tryptic peptide sequences of the pure protein a genomic DNA clone was obtained by polymerase chain reaction amplification. Its sequence contains an open reading frame of 1275 base pairs (EMBL nucleotide sequence data base, accession no. X97478X97478) encoding the SQR of R. capsulatus. The deduced polypeptide consists of 425 amino acid residues with a molecular mass of 47 kDa and a net charge of +9. The high similarity (72%)/identity (48%) between the N termini of the cyanobacterial and the bacterial enzyme was confirmed and extended. Both enzymes exhibit the FAD/NAD(P) binding betaalphabeta-fold (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). The complete sequence of the SQR from R. capsulatus shows further similarity to flavoproteins, in particular glutathione reductase and lipoamide dehydrogenase. The cloned sqr was expressed in Escherichia coli in a functional form.
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PMID:Sulfide-quinone reductase from Rhodobacter capsulatus. Purification, cloning, and expression. 909 26

A mutation leading to roseoflavin resistance and deregulated riboflavin biosynthesis was mapped in the genome of the riboflavin-overproducing Bacillus subtilis strains RB52 and RB50 at map position 147 degrees. The chromosomal location indicates that the deregulating mutation in RB52 and RB50 is an allele of the previously identified ribC mutation. We cloned the ribC gene and found that it encodes a putative 36-kDa protein. Surprisingly, RibC has significant sequence similarity to flavin kinases and FAD synthases from various other bacterial species. By comparing the deduced amino acid sequence of RibC from the wild-type parent strain of RB50 with the RibC sequence from the riboflavin-overexpressing RB50 mutant we identified a point mutation that resulted in a Gly to Ser exchange in the C-terminal region of the product.
Mol Gen Genet 1997 Mar 18
PMID:Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction. 910 93

Flavodoxin reductase from Escherichia coli is an FAD-containing oxidoreductase that transports electrons between flavodoxin or ferredoxin and NADPH. Together with flavodoxin, the enzyme is involved in the reductive activation of three E. coli enzymes: cobalamin-dependent methionine synthase, pyruvate formate lyase and anaerobic ribonucleotide reductase. An additional function for the oxidoreductase appears to be to protect the bacteria against oxygen radicals. The three-dimensional structure of flavodoxin reductase has been solved by multiple isomorphous replacement, and has been refined at 1.7 A to an R-value of 18.4% and Rfree 24.8%. The monomeric molecule contains one beta-sandwich FAD domain and an alpha/beta NADP domain. The overall structure is similar to other reductases of the NADP-ferredoxin reductase family in spite of the low sequence similarities within the family. Flavodoxin reductase lacks the loop which is involved in the binding of the adenosine moiety of FAD in other FAD binding enzymes of the superfamily but is missing in the FMN binding phthalate dioxygenase reductase. Instead of this loop, the adenine interacts with an extra tryptophan at the C terminus. The FAD in flavodoxin reductase has an unusual bent conformation with a hydrogen bond between the adenine and the isoalloxazine. This is probably the cause of the unusual spectrum of the enzyme. There is a pronounced cleft close to the isoalloxazine that appears to be well suited for binding of flavodoxin/ferredoxin. Two extra short strands of the NADP-binding domain probably act as an anchor point for the binding of flavodoxin.
J Mol Biol 1997 Apr 25
PMID:The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 A resolution. 914 48

Backbone-atom resonances have been assigned for both the substrate-free and the NADP+-complexed forms of UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme essential for bacterial cell-wall biosynthesis. NMR studies were performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 1HN and 15N atoms (94%), 315 13CO atoms (91%), 331 13C(alpha) atoms (95%), and 297 13C(beta) atoms (93%). For NADP+-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 1HN and 15N atoms (86%), 305 13CO atoms (88%), 310 13C(alpha) atoms (89%), and 269 13C(beta) atoms (84%). The strategies used for obtaining resonance assignments are described in detail. Information on the secondary structure in solution for both the substrate-free and NADP+-complexed forms of the enzyme has been derived both from 13C(alpha) and 13C(beta) chemical-shift deviations from random-coil values and from 1HN-1HN NOEs. These data are compared to X-ray crystallographic structures of substrate-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpyruvate (UNAGEP) substrate. NADP+ binding induces significant chemical-shift changes in residues both within the known UNAGEP and FAD binding pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP+ and UNAGEP utilize the same binding pocket and, furthermore, that the binding of NADP+ induces structural changes in MurB. Finally, many of the residues within the UNAGEP/NADP+ binding pocket were difficult to assign due to dynamic processes which weaken and/or broaden the respective resonances. Overall, our results are consistent with MurB having a flexible active site.
J Mol Biol 1997 Apr 18
PMID:Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes. 915 Apr 8

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated with the activation of the O2(-)-generating NADPH oxidase in a cell-free system. It is dependent upon NADPH, cytosolic factors, and amphiphiles (such as arachidonate), the same factors required for O2- generation. Both O2- generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH oxidase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mol. Biol. Biophys. (in press)]. In this report, the INT diaphorase activity of disrupted bovine polymorphonuclear neutrophils is shown to be resolved by DEAE-Sepharose chromatography into two fractions: an NADPH-cytochrome c reductase-containing fraction and a cytochrome b558-associated fraction. The diaphorase activity in the NADPH-cytochrome c reductase-containing portion is not dependent upon the presence of an amphiphile or phospholipid and is not associated with O2- generation. Upon incorporation into liposomes, the cytochrome b558-containing fraction demonstrates high O2- and INT reductase activities in the presence of cytosolic factors. Both O2- generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTPgammaS. Phenylarsine oxide inhibits both O2- generation and INT reductase activities when added prior to activation by SDS. With the cytochrome b-containing liposomes, the Km values (O2- formation) for NADPH and NADH are 27.2 microM and 810 microM, and for INT reductase the Km values are 27.5 microM and 1017 microM, respectively. Under anaerobic conditions and thus in the absence of O2- formation, the NADPH-dependent INT reductase activity does not change, indicating that the dye reduction is not due to its direct reduction by O2 anion but is an intrinsic property of the superoxide-generating NADPH oxidase. Cytochrome b558 is the essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The correlation of the activation and inhibition patterns for O2- generation and INT reduction by cytochrome b558 incorporated into artificial liposomes strongly indicates that the two activities are associated with the same membrane protein, cytochrome b558.
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PMID:Purified leukocyte cytochrome b558 incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity. 915 36

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 A 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.
J Mol Biol 1997 May 30
PMID:Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis. 919 5

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53,269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80 degrees C; ammonia-dependent reaction, 90 degrees C).
Mol Gen Genet 1997 May
PMID:Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1. 920 79

Control rats and diabetic animals injected with streptozotocin during the neonatal period were either maintained on a standard diet or given access to food supplemented with dehydroepiandrosterone (DHEA, 0.2%) for 11 days before sacrifice. In both control and diabetic rats, DHEA feeding augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in liver, but not so in either the parotid gland or pancreatic islets. DHEA lowered, in both control and diabetic rats, the ratio between D-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration of D-glucose, as well as the insulin concentration and insulin/glucose ratio in plasma. These findings support the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the otherwise unfavorable consequences of chronic hyperactivity.
Biochem Mol Med 1997 Jun
PMID:Effects of dehydroepiandrosterone in rats injected with streptozotocin during the neonatal period. 923


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