Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.     

Crystal structure of flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in two oxidation states

The crystal structures of the flavodoxin from Desulfovibrio desulfuricans ATCC 27774 have been determined and refined for both oxidized and semi-reduced forms to final crystallographic R-factors of 17.9% (0.8-0.205-nm resolution) and 19.4% (0.8-0.215-nm resolution) respectively. Native flavodoxin crystals were grown from ammonium sulfate with cell constants a = b = 9.59 nm, c=3.37nm (oxidized crystals) and they belong to space group P3(2)21. Semireduced crystals showed some changes in cell dimensions: a = b = 9.51 nm, c=3.35 nm. The three-dimensional structures are similar to other known flavodoxins and deviations are found essentially in the isoalloxazine ring environment. Conformational changes are observed between both redox states and a flip of the Gly61-Met62 peptide bond occurs upon one-electron reduction of the FMN group. These changes influence the redox potential of the oxidized/semiquinone couple. Modulation of the redox potentials is known to be related to the association constant of the FMN group to the protein. The flavodoxin from D. desulfuricans now studied has a large span between E2 (oxidized --> semiquinone) and E1 (semiquinone --> hydroquinone) redox potentials, both these values being substantially more positive within known flavodoxins. A comparison of their FMN environment was made in both oxidation states in order to correlate functional and structural differences.     

The primary structure of the split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveals an unusual type of diheme cytochrome c

The complete amino acid sequence of the unusual diheme split-Soret cytochrome c from the sulphate-reducing Desulfovibrio desulfuricans strain ATCC 27774 has been determined using classical chemical sequencing techniques and mass spectrometry. The 247-residue sequence shows almost no similarity with any other known diheme cytochrome c, but the heme-binding site of the protein is similar to that of the cytochromes c3 from the sulphate reducers. The cytochrome-c-like domain of the protein covers only the C-terminal part of the molecule, and there is evidence for at least one more domain containing four cysteine residues, which might bind another cofactor, possibly a non-heme iron-containing cluster. This domain is similar to a sequence fragment of the genome of Archaeoglobus fulgidus, which confirms the high conservation of the genes involved in sulfate reduction.     

Purification and characterization of the Rieske iron-sulfur protein from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

The previously detected Rieske iron-sulfur protein from the membranes of the thermoacidophile Sulfolobus acidocaldarius [Anemüller, S., et al. (1993) FEBS Lett. 318, 61-64] was purified to electrophoretic homogeneity and the N-terminal amino acids determined. The apparent molecular weight was estimated to be 32 kDa. The reduced protein displays a rhombic EPR spectrum with gxyz = 1.768, 1.895, 2.035. The average g-value of 1.902 is typical for nitrogen ligand-containing clusters. EPR spin quantification and the iron content indicate the presence of one [2Fe-2S] cluster. The purified protein displays ubiquinol cytochrome c reductase activity. The pH optimum of this reaction is temperature dependent and was determined to be pH 7 at 56 degrees C. The results presented in this study clearly prove that the Sulfolobus Rieske protein belongs to the family of the true Rieske iron-sulfur proteins.     

Physiologic studies with the sulfate-reducing bacterium Desulfovibrio desulfuricans: evaluation for use in a biofuel cell

The growth kinetics of the sulfate-reducing bacteria Desulfovibrio desulfuricans Essex 6 was investigated under various conditions for potential use in a microbial fuel cell that recovers electrons generated from the reduction of sulfate to hydrogen sulfide. Hydrogen sulfide was found to inhibit growth and decrease both the growth yields and the sulfate-specific reduction rate. Hydrogen sulfide inhibition was direct, reversible, and not due to limitation by iron deficiency. A high initial lactate concentration also retarded bacterial growth, reduced the specific sulfate reduction rates, and gave variable biomass growth yields. This effect resulted from a bottleneck in the lactate oxidation pathway which induced the production of the secondary product butanol. The use of pyruvate as a carbon source was more advantageous than lactate in terms of growth rate and biomass growth yields, with only a slight decrease in the rate of specific sulfate reduction. For equal biomass, a slightly higher current density was generated from lactate than pyruvate, but pyruvate required nearly 40% less sulfate.     

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.     

Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana

Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).     

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58-60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C alpha-distance 1.2 A). However, this peroxidase includes a Mn2+ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn2+. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae

Cytochrome oxidase (COX) is considered to integrate in a single enzyme two consecutive mechanistically different redox activities--oxidase and peroxidase--that can be catalyzed elsewhere by separate hemoproteins. From the viewpoint of energy transduction, the enzyme is essentially a proton pumping peroxidase with a built-in auxiliary eu-oxidase module that activates oxygen and prepares in situ H2O2, a thermodynamically efficient but potentially hazardous electron acceptor for the proton pumping peroxidase. The eu-oxidase and peroxidase phases of the catalytic cycle may be performed by different structural states of COX. Resolution of the proton pumping peroxidase activity of COX and identification of individual charge translocation steps inherent in this reaction are discussed, as well as the specific role of the two input proton channels in proton translocation.     

Identification and developmental characterization of a novel Y-box protein from Drosophila melanogaster

The Y-box proteins are a family of highly conserved nucleic acid binding proteins which are conserved from bacteria to human. In this report we have identified a new member of this family from Drosophila melanogaster. Degenerate-PCR was used to identify a conserved region within the highly conserved cold-shock domain (CSD) of Y-box proteins. Subsequently, the cDNA for this gene was sequenced, and the identified open reading frame was named ypsilon schachtel (yps). The expression pattern of yps indicates that this gene is expressed throughout development with the highest level of expression found in adult flies. In situ hybridization shows that the yps mRNA is maternally loaded into the egg cytoplasm. In addition, there appears to be expression of yps mRNA in mesodermal tissue during embryogenesis. YPS, while containing a conserved CSD, is novel in that it completely lacks the alternating acidic and basic regions found in the C-terminus of the other vertebrate eukaryotic Y-box proteins. The CSD of yps was purified and gel-shift analysis showed that this domain can interact with RNA. We predict that YPS would be an RNA-binding protein due to these results and the motifs which have been identified within the amino acid sequence.     

Growth of a human intestinal Desulfovibrio desulfuricans in continuous cultures containing defined populations of saccharolytic and amino acid fermenting bacteria

Ecological and physiological effects of the sulphate-reducing bacterium (SRB) Desulfovibrio desulfuricans on other intestinal organisms were investigated in anaerobic chemostats (dilution rate approximately 0.2 h-1). Reproducible defined bacterial communities were used in these experiments, comprising 14 different saccharolytic and amino acid fermenting species: Bifidobacterium longum, Bif. adolescentis, Bif. pseudolongum, Bif. infantis, Bacteroides thetaiotaomicron, Bact. vulgatus, Lactobacillus acidophilus, Enterococcus faecalis, Ent. faecium, Escherichia coli, Clostridium perfringens, Cl. butyricum, Cl. innocuum, Cl. bifermentans. Lactobacillus and Cl. bifermentans populations never rose above minimum detection limits (log10 2.0 and 4.0, respectively) under the experimental conditions employed in these studies. Inclusion of Des. desulfuricans in bacterial cultures (c. log10 8.4 viable cells ml-1) resulted in marked reductions (i.e. greater than 1 log) in planktonic cell population densities of several species, particularly Bif. longum, Cl. perfringens and Bif. pseudolongum. The two bacteroides species were unaffected by Des. desulfuricans, while numbers of Cl. butyricum increased. Extensive wall growth developed in the SRB culture, consisting mainly of Des. desulfuricans (log10 9.2 viable cells ml-1), Bact. thetaiotaomicron and Bact. vulgatus, with lesser numbers of facultative anaerobes, Cl. perfringens and Bif. longum. Wall growth was associated with a reduction in planktonic cell mass and increased acid production by the cultures. Chemotaxonomic study of chemostat microbiotas, on the basis of cellular fatty acid methyl ester (FAME) analyses, showed the existence of characteristic bacteroides (C15) and bifidobacterial (C18) markers, but desulfovibrio markers (i-C15:0, C16:0, i-C17:1) could be identified. The metabolic activities of saccharolytic organisms were altered in the SRB chemostat, including synthesis of a number of hydrolytic enzymes involved in carbohydrate breakdown, such as alpha-galactosidase, alpha-glucosidase and beta-galactosidase, together with several mucinolytic enzymes. High concentrations of sulphide (8.2 mmol 1-1) were detected in the SRB chemostat, suggesting that this metabolite may have been inhibitory to some species. Saccharolytic organisms growing in the SRB fermenter utilized more starch, but less galactose-containing polymers, which correlated with the observed glycosidase activities. Profound differences were also recorded with respect to fermentation product formation in the chemostats, where a major switch to acetate production occurred in the SRB culture, with concomitant reductions in propionate, butyrate and lactate, which is an important electron donor for desulfovibrios.     

Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S]+) from axial to rhombic symmetry.     

Isolation and characterization of a novel inositol hexakisphosphate binding protein from mammalian cell cytosol

Inositol hexakisphosphate (IP6) is present in most mammalian cells, although its intracellular function is as yet undefined. We find that the total protein fraction from bovine brain cytosol contains a significant level of specific binding for IP6 precipitable with 40% saturated ammonium sulfate. A protein complex has been isolated from this fraction that specifically binds IP6 and is purified about 500-fold over the cytosol. The IP6 binding protein (IP6BP) chromatographs as a single peak of binding activity on a gel exclusion column, with a Stokes radius equivalent to 266 +/- 14 kDa. The IP6BP is a heterooligomeric complex composed of a number of subunits with molecular weights varying from 23,000 to 60,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Scatchard analyses of IP6 binding of both the crude ammonium sulfate fraction and the purified complex show the presence of a similar high-affinity binding site (Kd approximately 6.0 nM). Bmax for the purified fraction is 1.8 nmol of IP6/mg of protein or 0.48 mol of IP6 bound/mol of complex. Other inositol polyphosphates, such as inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate, are poor competitors for IP6 binding to the purified complex. The purification scheme, when applied to a rat liver cytosol fraction, yields a similar IP6BP. This complex has an apparent size of 512,000 using gel exclusion chromatography and contains an additional protein band with M(r) = 97,000 by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of a novel redox protein by rational design: conversion of a disulfide bridge into a mononuclear iron-sulfur center

A mononuclear iron-sulfur center, capable of reversible electron transfer, has been introduced into thioredoxin, a protein devoid of such sites, using an automated, structure-based design algorithm. One of the sites predicted by the Dezymer computer program to introduce a tetrahedral tetrathiolate iron center included the intrinsic Cys32-Cys35 disulfide of wild-type thioredoxin and two additional mutants, Trp28Cys and Ile75Cys, thereby converting a disulfide into a metal-based redox center. This designed protein forms a 1:1 monomeric complex with FeIII, whose electronic absorption and EPR spectra closely resemble those of the rubredoxins, as intended. CoII spectra provided further confirmation of tetrahedral tetrathiolate metal coordination. The designed protein is capable of undergoing successive cycles of oxidation and reduction. The computer-generated design only took into account the geometry of the primary coordination shell around the metal. We have therefore demonstrated that simple geometrical considerations can be sufficient to reproduce the dominant electronic structure and reactivity of a simple metal-based redox center.     

Electrophysiological characterization of human distal colon epithelium isolated using a novel technique

Electrophysiological studies of human colonic epithelia traditionally have been hampered by the lack of tissue availability and by poor tissue quality. Human colonic epithelium is usually obtained surgically from individuals with underlying disease, while surgery itself can injure or alter the resected tissue. As a result, a wide range in electrophysiological parameters is reported in previous studies of human colonic epithelium. Such factors may also account for differences in measurements between humans and the few other species studied. We therefore devised a novel and rapid endoscopic technique, endoscopic mucosal resection (EMR), that allows for the removal and study of intestinal mucosal epithelium from normal volunteers. Using EMR we rapidly (7.2+/-2.4 min) isolated surgical-sized epithelial sheets from the distal colon (1.4+/-0.4 by 1.3+/-0.4 cm) that were readily mounted in a 0.64-cm2 Ussing chamber. We observed stable resistance (289+/-30 omega cm2), potential difference (1.6+/-0.6 mV), and I(SC)(24+/-9 microA/cm2) for at least 90 min, after which all experiments were terminated. Exposure to carbachol increased I(SC)2.2+/-0.5-fold, while forskolin increased I(SC) 4.4+/-0.5-fold. These data show that the electrophysiological characteristics of the human distal colon removed by EMR more closely approximate values reported for other mammals than when removed using other techniques. Thus EMR represents a significant advance over traditional techniques for isolating human tissues and will increase the availability of this tissue for future studies.     

Genetic characterization of an oral treponeme isolated from human subgingival plaque

INTRODUCTION: Since many benign and malignant pathologic conditions can appear as solitary pulmonary nodules, to establish nodule nature is always necessary for correct patient management. Recently, some authors have demonstrated the effectiveness of incremental dynamic CT in distinguishing cancerous from noncancerous lesions. The purpose of this work is to report our personal experience in this field. MATERIAL AND METHODS: We reviewed the incremental dynamic CT scans of 21 patients with a solitary pulmonary nodule < 3.5 cm phi without any calcifications, cavities and fat--namely, 15 carcinomas, 3 granulomas, 2 hamartomas, 1 abscess. Lesion density was evaluated before and 30 s, 1, 2, 3 and 5 min after contrast agent administration; we used a circular region of interest consisting of the central portion of the nodule in all cases and of 60-70% of its area in most cases. We subdivided the nodules into two groups, according to their enhancement: the nodules with > 20 HU and those with < 20 HU. All the lesions were submitted to surgery and histologic studies. RESULTS: Fourteen of 15 carcinomas and one hamartoma had contrast enhancement > 20 HU; an abscess exhibited marked ring-shaped contrast enhancement (positive predictive value: 87%). One carcinoma, three granulomas and one hamartoma had no contrast enhancement, or else it was < 20 HU (negative predictive value: 80%). DISCUSSION: Recently, some authors have demonstrated that malignant nodules, studied with incremental dynamic CT, have higher contrast enhancement than benign nodules. A value > 20 HU is a good predictor of malignancy (positive predictive value: 90%) and, conversely, a value < 20 HU is an unquestionable sign of benignity (negative predictive value: 100%). Our findings confirm the positive predictive value of enhancement > 20 HU, but not its negative predictive value because we found a malignant nodule without contrast enhancement. CONCLUSIONS: Incremental dynamic CT is an effective indicator of solitary pulmonary nodule nature, but its predictive value is not absolute and therefore this technique should be integrated with biopsy in the cases which are clinically or radiologically suspicious.