|
|
|
|
|
|
Flavin Mononucleotide-Binding Flavoprotein Family in the Domain Archaea. Yan-Huai R. Ding, 2004.The protein (AfpA, for archaeoflavoprotein) encoded by AF1518 in the genome of Archaeoglobus fulgidus was produced in Escherichia coli and characterized . AfpA was found to be a homodimer with a native molecular mass of 43 kDa and containing two noncovalently bound flavin mononucleotides (FMNs) . The cell extract of A . fulgidus catalyzed the CO-dependent reduction of AfpA that was stimulated by the addition of ferredoxin . Ferredoxin was found to be a direct electron donor to purified AfpA, whereas rubredoxin was unable to substitute . Neither NADH nor NADPH was an electron donor . Ferricyanide, 2,6-dichlorophenolindophenol, several quinones, ferric citrate, bovine cytochrome c, and O2 accepted electrons from reduced AfpA, whereas coenzyme F420 did not . The rate of cytochrome c reduction was enhanced in the presence of O2 suggesting that superoxide is a product of the interaction of reduced AfpA with O2 . Although AF1518 was previously annotated as encoding a decarboxylase involved in coenzyme A biosynthesis, the results establish that AfpA is an electron carrier protein with ferredoxin as the physiological electron donor . The genomes of several diverse Archaea contained afpA homologs clustered with open reading frames annotated as homologs of genes encoding reductases involved in the oxidative stress response of anaerobes from the domain Bacteria . A potential role for AfpA in coupling electron flow from ferredoxin to the putative reductases is discussed . A search of the databases suggests that AfpA is the prototype of a previously unrecognized flavoprotein family unique to the domain Archaea for which the name archaeoflavoprotein is proposed . Phylogenetic and Ecological Analysis of Novel Marine Stramenopiles. Ramon Massana, 2004.Culture-independent molecular analyses of open-sea microorganisms have revealed the existence and apparent abundance of novel eukaryotic lineages, opening new avenues for phylogenetic, evolutionary, and ecological research . Novel marine stramenopiles, identified by 18S ribosomal DNA sequences within the basal part of the stramenopile radiation but unrelated to any previously known group, constituted one of the most important novel lineages in these open-sea samples . Here we carry out a comparative analysis of novel stramenopiles, including new sequences from coastal genetic libraries presented here and sequences from recent reports from the open ocean and marine anoxic sites . Novel stramenopiles were found in all major habitats, generally accounting for a significant proportion of clones in genetic libraries . Phylogenetic analyses indicated the existence of 12 independent clusters . Some of these were restricted to anoxic or deep-sea environments, but the majority were typical components of coastal and open-sea waters . We specifically identified four clusters that were well represented in most marine surface waters (together they accounted for 74% of the novel stramenopile clones) and are the obvious targets for future research . Many sequences were retrieved from geographically distant regions, indicating that some organisms were cosmopolitan . Our study expands our knowledge on the phylogenetic diversity and distribution of novel marine stramenopiles and confirms that they are fundamental members of the marine eukaryotic picoplankton . Sugar Transport through Maltoporin of Escherichia coli: Role of the Greasy Slide. Patrick Van Gelder, 2002.The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding . To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction . The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis . Crystallographic analysis of the W74A mutant showed no perturbation of the structure . All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues . Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel . Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition . The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel . Novel Carbohydrate-Binding Module of ß-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp . Strain XY-234. Fumiyoshi Okazaki, 2002.A ß-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp . strain XY-234, has been cloned and sequenced . txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da . The domain structure of the ß-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus . The recombinant TxyA hydrolyzed ß-1,3-xylan but not other polysaccharides such as ß-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or ß-1,4-mannan . TxyA was capable of binding specifically to ß-1,3-xylan . The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469 . Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to ß-1,3-xylan were 4.2 µM and 18.2 µmol/g of ß-1,3-xylan, respectively . Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of ß-1,3-xylan . Changes in Escherichia coli rRNA Promoter Activity Correlate with Changes in Initiating Nucleoside Triphosphate and Guanosine 5' Diphosphate 3'-Diphosphate Concentrations after Induction of Feedback Control of Ribosome Synthesis. David A. Schneider, 2003.rRNA synthesis is the rate-limiting step in ribosome synthesis in Escherichia coli . Its regulation has been described in terms of a negative-feedback control loop in which rRNA promoter activity responds to the amount of translation . The feedback nature of this control system was demonstrated previously by artificially changing ribosome synthesis rates and observing responses of rRNA promoters . However, it has not been demonstrated previously that the initiating nucleoside triphosphate (iNTP) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), the molecular effectors responsible for controlling rRNA promoters in response to changes in the nutritional environment, are responsible for altering rRNA promoter activities under these feedback conditions . Here, we show that most feedback situations result in changes in the concentrations of both the iNTP and ppGpp and that the directions of these changes are consistent with a role for these two small-molecule regulators in feedback control of rRNA synthesis . In contrast, we observed no change in the level of DNA supercoiling under the feedback conditions examined .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
