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

Electron-transfer flavoprotein from Methylophilus W3A1 has been crystallized using the sitting drop vapour diffusion technique. Hexagonal crystals, suitable for high-resolution structure determination grow to approximately 0.7 mm x 0.7 mm x 0.5 mm in size and diffract to at least 2.2 A. The space group is either P6(1) or P6(5) with unit cell dimensions a,b = 119.1 A and c = 85.6 A.
J Mol Biol 1994 Jul 15
PMID:Crystallization and preliminary crystallographic investigation of electron-transfer flavoprotein from the bacterium Methylophilus W3A1. 802 9

An enzyme catalyzing sulfide quinone oxido-reduction (E.C.1.8.5.'.; SQR) has been purified in an active form, from thylakoids of the cyanobacterium Oscillatoria limnetica. It is composed of a single polypeptide of about 57 kDa. The catalytic activity of the purified enzyme is similar to the membrane-bound form in its kinetic parameters: apparent Km for sulfide equals 8 microM; Vmax of 100-150 mumol of plastoquinone-1 reduced/mg protein/h; quinone-substrate specificity; differential sensitivity to quinone analog inhibitors, the most potent of which being aurachin C (I50 = 7 nM), and specific inducibility by sulfide. Taken together, they suggest that the purified SQR is the enzyme catalyzing anoxygenic photosynthesis in cyanobacteria. The UV and visible absorption and fluorescence spectra of the purified SQR are typical of a flavoprotein. Both the absorption and fluorescence intensities are reduced by sulfide. The SQR activity is inhibited by KCN, a flavoprotein inhibitor. We have sequenced so far 29 amino acid residues of the SQR NH2 terminus and found that from the second residue, this sequence contains the highly conserved fingerprint of the NAD/FAD-binding domain of many NAD/FAD-binding enzymes (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). This suggests that the SQR enzyme is a flavoprotein which contains binding sites for sulfide and quinone and that the electron transfer between the two is mediated by FAD.
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PMID:Purification and characterization of sulfide-quinone reductase, a novel enzyme driving anoxygenic photosynthesis in Oscillatoria limnetica. 811 8

The LIM2 gene may play a major role in lens fiber cell structure or communication, and thus cataractogenesis. A human cDNA encoding the corresponding lens fiber cell intrinsic protein MP19 has been previously isolated and characterized. This cDNA had been mapped to human chromosome 19. We have independently confirmed this assignment and fine mapped it to 19q13.4. The position of the LIM2 gene appears to be within 40 kb of the electron transport flavoprotein gene (ETFB) as a cosmid containing sequences from both genes has been identified.
Somat Cell Mol Genet 1994 Jan
PMID:Assignment of the human lens fiber cell MP19 gene (LIM2) to chromosome 19q13.4, and adjacent to ETFB. 819 79

The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.
J Mol Biol 1993 Apr 20
PMID:Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties. 848 1

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of L- and D-lactate to pyruvate catalysed by L-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and D-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate- pyruvate+ mutants in a cyb2 genetic background. Two of them were devoid of D-LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on D-, L-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein L-gulono-gamma-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of L-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.
Mol Gen Genet 1993 Apr
PMID:Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase. 849 99

We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH). Their half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and < 5 min in glucose (YPD medium). This is a rare example in yeast in which the half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and < 5 min in glucose (YPD medium). This is a rare example in yeast in which the half-life of an mRNA can be controlled by manipulating external conditions. In our current studies, a series of Ip transcripts with internal deletions as well as chimeric transcripts with heterologous sequences (internally or at the ends) have been examined, and we established that the 5'-untranslated region (5' UTR) of the Ip mRNA contains a major determinant controlling its differential turnover in YPG and YPD. Furthermore, the 5' exonuclease encoded by the XRN1 gene is required for the rapid degradation of the Ip and Fp mRNAs upon the addition of glucose. In the presence of cycloheximide the nucleolytic degradation of the Ip mRNA can be slowed down by stalled ribosomes to allow the identification of intermediates. Such intermediates have lost their 5' ends but still retain their 3' UTRs. If protein synthesis is inhibited at an early initiation step by the use of a prt1 mutation (affecting the initiation factor eIF3), the Ip and Fp mRNAs are very rapidly degraded even in YPG. Significantly, the arrest of translation by the introduction of a stable hairpin loop just upstream of the initiation codon does not alter the differential stability of the transcript in YPG and YPD. These observations suggest that a signaling pathway exists in which the external carbon source can control the turnover of mRNAs of specific mitochondrial proteins. Factors must be present that control either the activity or more likely the access of a nuclease to the select mRNAs. As a result, we propose that a competition between initiation of translation and nuclease action at the 5' end of the transcript determines the half-life of the Ip mRNA.
Mol Biol Cell 1995 Sep
PMID:Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role. 853 11

In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at -160 degrees C was applied. The structure of the native enzyme to 2.8 A resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid. The Cys42-sulfenic acid atoms C alpha-C beta-S gamma-O roughly define a planar arrangement which is stacked parallel to the si face of the FAD isoalloxazine and positions the sulfenyl oxygen atom only 3.3 A from FAD-C4A. His10-N epsilon 2 contributes a hydrogen bond to the sulfenic acid oxygen, at a distance of 3.2 A. Although one oxygen atom (OX1) of the non-native Cys42-sulfonic acid derivative identified in the earlier wild-type peroxidase structure was taken to represent the native Cys42-sulfenic acid oxygen [Stehle, T., Ahmed, S. A., Claiborne, A., & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344], this structure shows that the sulfenic acid oxygen does not occupy this position, nor is it hydrogen-bonded to Cys42-N as was OX1. Comparison of the native Cys42-sulfenic acid structure with that of two-electron reduced glutathione reductase provides an insight into the sulfenic acid FAD charge-transfer interaction observed with both wild-type and His10 mutant peroxidases. A model of the E.NADH intermediate recently observed in stopped-flow analyses of the enzyme [Crane, E. J., III, Parsonage, D., Poole, L. B., & Claiborne, A. (1995) Biochemistry 34, 14114-14124] has also been generated to assist in analyzing the chemical mechanism of sulfenic acid reduction.
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PMID:Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 A resolution. 875 56

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

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 key step in plant photorespiration, the oxidation of glycolate to glyoxylate, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). The three-dimensional structure of this alpha/beta barrel protein has been refined to 2 A resolution (Lindqvist Y. 1989. J Mol Biol 209:151-166). FMN dependent glycolate oxidase is a member of the family of alpha-hydroxy acid oxidases. Here we describe the crystallization and structure determination of two inhibitor complexes of the enzyme, TKP (3-Decyl-2,5-dioxo-4-hydroxy-3-pyrroline) and TACA (4-Carboxy-5-(1-pentyl)hexylsulfanyl-1,2,3-triazole). The structure of the TACA complex has been refined to 2.6 A resolution and the TKP complex, solved with molecular replacement, to 2.2 A resolution. The Rfree for the TACA and TKP complexes are 24.2 and 25.1%, respectively. The overall structures are very similar to the unliganded holoenzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced flexible loop in the TKP complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different Ki values for the inhibitors, 16 nM for TACA and 4.8 microM for TKP. Implications of the structures of these enzyme-inhibitor complexes for the model for substrate binding and catalysis proposed from the holo-enzyme structure are discussed.
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PMID:Three-dimensional structures of glycolate oxidase with bound active-site inhibitors. 914 71


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