bluuebunny/crossref_metadata_2025 huggingface dataset:
Methanobacterium thermoautotrophicum encodes two multisubunit membrane‐bound [NiFe] hydrogenases
Authors: Tersteegen Adrian , Hedderich Reiner
Published: March 2003

Two gene groups, designated energy converting hydrogenase A (eha) and energy converting hydrogenase B (ehb), each encoding a putative multisubunit membrane‐bound [NiFe] hydrogenase, were identified in the genome of Methanobacterium thermoautotrophicum. The length of the transcription units was determined using reverse transcription (RT)‐PCR. The eha operon (12.5 kb) and the ehb operon (9.6 kb) were found to be composed of 20 and 17 open reading frames, respectively. Competitive RT‐PCR was used to compare the amounts of eha and ehb transcripts with the amounts of transcripts of genes encoding the M. thermoautotrophicum catabolic enzymes cyclohydrolase (mch) and a subunit of heterodisulfide reductase (hdrC). In cells grown under conditions in which H2 was nonlimiting, the eha transcripts were 250‐fold and 125‐fold less abundant and the ehb transcripts were approximately sixfold and threefold less abundant than the hdrC and mch transcripts, respectively. In cells grown under H2 limitation, the amounts of eha and ehb transcripts were about threefold higher than in cells grown with sufficient H2 when compared to the amounts of hdrC and mch transcripts. Sequence analysis of the deduced proteins indicated that the eha and ehb operons each encode a [NiFe] hydrogenase large subunit, a [NiFe] hydrogenase small subunit, and two conserved integral membrane proteins. These proteins show high sequence similarity to subunits of the Ech hydrogenase from Methanosarcina barkeri, Escherichia coli hydrogenases 3 and 4, and CO‐induced hydrogenase from Rhodospirillum rubrum, all of which form a distinct group of multisubunit membrane‐bound [NiFe] hydrogenases and show high sequence similarity to the energy‐conserving NADH:quinone oxidoreductase (complex I) from various organisms. In addition to these four subunits, the eha operon encodes a 6[4Fe–4S] polyferredoxin, a 10[4F–4S] polyferredoxin, four nonconserved hydrophilic subunits, and 10 nonconserved integral membrane proteins; the ehb operon encodes a 2[4Fe–4S] ferredoxin, a 14[4Fe–4S] polyferredoxin, two nonconserved hydrophilic subunits, and nine nonconserved integral membrane proteins. A function of these putative membrane‐bound [NiFe] hydrogenases as proton pumps involved in endergonic reactions, such as the synthesis of formylmethanofuran from CO2, H2 and methanofuran, is discussed.
The effects of several nucleotides on the molecular state and catalytic activity of Thiobacillus novellus cytochrome c oxidase
Authors: Shoji Kazuo , Tanigawa Minoru , Hori Kiyoaki , Tomozawa Yukiko , Yamanaka Tateo
Published: March 2003

The catalytic activity and molecular aspects of Thiobacillus novellus cytpchrome c oxidase were affected by ATP. The steady‐state kinetics in the oxidation of ferrocytochrome c by the oxidase varied with the presence or absence of ATP; the [S]–v curve of the reaction was sigmoid in the absence of ATP whereas it was a Michaelis–Menten‐type hyperbola in the presence of 700 µm ATP. The oxidase was a dimer of the minimal structural subunit consisting of one molecule each of two subunits in the presence of Tween 20 and in the absence of ATP. The dimer dissociated into monomers in the presence of 700 µm ATP. The trough at 452 nm seen in the second derivative absorption spectrum of the CO compound of the oxidase in the absence of ATP, a characteristic of the cytochrome a component of cytochrome aa3, dissappeared in the presence of 700 µm ATP. However, ADP, AMP, GTP, CTP and UTP had little affect on both the [S]–v curve and the molecular mass of the oxidase when used in place of ATP.
Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme
Authors: Pessione Enrica , Divari Sara , Griva Ersilia , Cavaletto Maria , Rossi Gian L. , Gilardi Gianfranco , Giunta Carlo
Published: March 2003

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron–sulfur cluster of the type [2Fe–2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2,6‐dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N‐terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.
Formation of the neuromuscular junction
Authors: Hoch Werner
Published: March 2003

Synapses are essential relay stations for the transmission of information between neurones and other cells. An ordered and tightly regulated formation of these structures is crucial for the functioning of the nervous system. The induction of the intensively studied synapse between nerve and muscle is initiated by the binding of neurone‐specific isoforms of the basal membrane protein agrin to receptors on the surface of myotubes. Agrin activates a receptor complex that includes the muscle‐specific kinase and most likely additional, yet to be identified, components. Receptor activation leads to the aggregation of acetylcholine receptors (AChR) and other proteins of the postsynaptic apparatus. This activation process has unique features which distinguish it from other receptor tyrosine kinases. In particular, the autophosphorylation of the kinase domain, which usually induces the recruitment of adaptor and signalling molecules, is not sufficient for AChR aggregation. Apparently, interactions of the extracellular domain with unknown components are also required for this process. Agrin binds to a second protein complex on the muscle surface known as the dystrophin‐associated glycoprotein complex. This binding forms one end of a molecular link between the extracellular matrix and the cytoskeleton. While many components of the machinery triggering postsynaptic differentiation have now been identified, our picture of the molecular pathway causing the redistibution of synaptic proteins is still incomplete.
Inhibition of phosphodiesterase/pyrophosphatase activity of PC‐1 by its association with glycosaminoglycans
Authors: Hosoda Nao , Hoshino Shin‐ichi , Kanda Yasunari , Katada Toshiaki
Published: March 2003

PC‐1 is a type II membrane‐bound glycoprotein consisting of a short N‐terminal cytoplasmic domain and a large C‐terminal extracellular domain, which contains phosphodiesterase/pyrophosphatase activity. When Jurkat T cells were cultured with dibutyryl cAMP, the membrane‐bound PC‐1 and its soluble form were induced. They were purified as a homodimer of a 130 kDa peptide and a 120 kDa monomer, respectively, and the same two forms could also be obtained from COS‐7 cells that had been transfected with PC‐1 cDNA. The membrane‐bound and soluble forms of PC‐1 were indistinguishable from each other in terms of their enzyme kinetics and N‐glycosylated moieties. Thus, the enzymatically active and fully glycosylated form of soluble PC‐1 was utilized to search for its interacting molecules. The phosphodiesterase/pyrophosphatase activity of PC‐1 was competitively inhibited by glycosaminoglycans, such as heparin and heparan sulfate, which are the major components of the extracellular matrix. PC‐1 was capable of binding to heparin–Sepharose and the binding was inhibited in the presence of the enzyme substrate, ATP or its nonhydrolyzable analog. The enzyme activity of PC‐1 itself, however, was not required for the binding to heparin–Sepharose. These results suggest that PC‐1 might function as an adhesion molecule independent of its enzyme activity to associate with glycosaminoglycans in the extracellular matrix.
Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG
Authors: Miège Christine , Maréchal Eric , Shimojima Mie , Awai Koichiro , Block Maryse A. , Ohta Hiroyuki , Takamiya Ken‐ichiro , Douce Roland , Joyard Jacques
Published: March 2003

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both ‘prokaryotic’ and ‘eukaryotic’ MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N‐ethylmaleimide and o‐phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic α helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of ≈ 45 kDa. In addition, inactivation kinetics after γ‐ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95–100 kDa, indicating that they are probably active as homodimers made of two 45‐kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.
An RNA polymerase III transcription unit located in the upstream control regions of the human proliferating‐cell nucleolar protein p120 gene is transcribed in vitro and in vivo
Authors: Lowe David , Henning Dale , Reddy Ram
Published: March 2003

An RNA polymerase III (Pol III) transcription unit containing homology to highly repeated Alu sequences was identified in the upstream flanking sequences of the gene for the human proliferating‐cell nucleolar antigen p120. When transcribed in vitro, this Pol III unit produced three RNA transcripts, designated by nucleotide length as T150, T385 and T635; RNA transcript T635 was the most abundant accounting for over 90%. The transcription initiates at nucleotide −729 of the human p120 promoter and proceeds in the opposite orientation to the p120 gene transcription. Northern blot analysis and cDNA cloning followed by sequencing showed the presence of the T635 RNA in HeLa cells, indicating that this Pol III transcription unit is functional and transcribed in vivo. Disruption of this Pol III transcription unit by deletion of the Box A residues (−733 to −744) resulted in a sixfold reduction of the p120 gene transcription. A possible role for this Pol III transcription unit in p120 gene transcription is discussed.
Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure
Authors: De Felice Fernanda G. , Soares Vanessa C. , Ferreira Sérgio T.
Published: March 2003

The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size‐exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure‐induced subunit dissociation. Size‐exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure‐dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far‐UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K+, Mg2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.
Characterization and DNA‐binding properties of GRF, a novel monomeric binding orphan receptor related to GCNF and βFTZ‐F1
Authors: Charles Jean‐Philippe , Shinoda Tetsuro , Chinzei Yasuo
Published: March 2003

A PCR approach has been used to isolate, from Bombyx mori, a cDNA encoding a novel orphan receptor (GRF) that is most closely related to BombyxβFTZ‐F1 and to the vertebrate germ cell nuclear factor. The major GRF mRNA is detected in most tissues as an 8‐kb transcript whose amount follows the circulating ecdysteroid concentration with a delay. The expression pattern of GRF is similar to that of the Bombyx homologue of the Drosophila early‐late gene DHR3, and precedes that of βFTZ‐F1 in all stages and tissues examined. The GRF protein is thus likely to be required in many tissues, but in a temporally restricted manner suggesting that GRF has a well‐defined function in the ecdysteroid‐induced transcription cascade. The GRF protein binds in vitro to a single oestrogen receptor half‐site AGGTCA preceded by a 5′‐TCA extension, and is therefore a potential co‐regulator of the orphan receptors βFTZ‐F1 and DHR39.
Structural studies of the O‐antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross‐reacting with Shigella dysenteriae type 3
Authors: Linnerborg Malin , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigen polysaccharide from Escherichia coli O164 has been determined. Nuclear magnetic resonance spectroscopy together with component and methylation analyses of lipid free polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity NMR experiments. It is concluded that the polysaccharide is composed of a pentasaccharide repeating unit with the following structure:

Matrix assisted laser desorption ionization mass spectrometry (MALDI‐MS) was performed on intact lipopolysaccharide and from the resulting molecular mass, the O‐antigen part was estimated to contain approximately 24 repeating units. The nature of the previously reported cross‐reactivity of this O‐antigen to those of Escherichia coli O124 and Shigella dysenteriae type 3 is discussed.
Self‐assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus
Authors: Domingo Gonzalo J. , Chauhan Hitesh J. , Lessard Ivan A. D. , Fuller Christopher , Perham Richard N.
Published: March 2003

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over‐expressed in Escherichia coli. Titrations of the icosahedral (60‐mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, α2β2) and dihydrolipoyl dehydrogenase (E3, α2) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit‐binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1–E2 or E3–E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit‐binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active‐site coupling.
Solution structure of a recombinant mouse major urinary protein
Authors: Lücke Christian , Franzoni Lorella , Abbate Francesco , Löhr Frank , Ferrari Elena , Sorbi Robert T. , Rüterjans Heinz , Spisni Alberto
Published: March 2003

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism.

In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild‐type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently.

Here, we describe the refined high‐resolution three‐dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE‐derived distance restraints. A comparison with the crystal structure of the wild‐type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology.

The secondary structure of the protein consists of eight antiparallel β‐strands forming a single β‐sheet and an α‐helix in the C‐terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the β‐strands. The tertiary fold of the β‐sheet creates a β‐barrel, common to all members of the lipocalin superfamily. The shape of the β‐barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.
Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix
Authors: Tachibana Akira , Yano Yoshihisa , Otani Shuzo , Nomura Norimichi , Sako Yoshihiko , Taniguchi Makoto
Published: March 2003

Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates. Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20–C25 diether lipids in addition to C20–C20 diether lipids commonly occurring in archaea [Tachibana, A. (1994) FEBS Lett.341, 291–294]. Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25–C25 diether lipids, not the usual C20‐containing lipids [Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y. & Koga, Y. (1999) Biochim. Biophys. Acta1436, 426–436]. In this report, we describe the isoloation from A. pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase. The protein encoded by fgs was expressed in Escherichia coli as a glutathione S‐transferase fusion protein and produced FGPP as a final product. Phylogenetic analysis of fgs with other prenyltransferases revealed that the short‐chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III. fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity.
NADP+‐dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1
Authors: Di Fraia Raffaela , Wilquet Valerie , Ciardiello M. Antonietta , Carratore Vito , Antignani Antonella , Camardella Laura , Glansdorff Nicolas , di Prisco Guido
Published: March 2003

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+‐dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme‐binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five‐residue insertion in a loop between two α‐helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10–30 °C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2‐oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold‐active enzymes.
The major site of the Pti1 kinase phosphorylated by the Pto kinase is located in the activation domain and is required for Pto–Pti1 physical interaction
Authors: Sessa Guido , D’Ascenzo Mark , Martin Gregory B.
Published: March 2003

The Pto and Pti1 serine/threonine protein kinases are key components of the signaling pathway leading to speck disease resistance in tomato. The two kinases physically interact in the yeast two‐hybrid system, and Pto specifically phosphorylates Pti1 in vitro. In this study, we identified and characterized the major Pti1 site phosphorylated by Pto. Pto was expressed in Escherichia coli as a maltose‐binding fusion protein (MBP‐Pto), and used to phosphorylate in vitro a kinase deficient Pti1 protein fused to glutathione S‐transferase (GST‐Pti1[K96N]). The major phosphopeptide derived from trypsin digestion of phosphorylated GST‐Pti1(K96N) was partially purified by reverse‐phase HPLC and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230–234. By phosphoamino acid analysis, Thr233 was determined to be the phosphorylation site of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phosphorylation by MBP‐Pto and Pti1 autophosphorylation, providing evidence that the same Pti1 site is involved in the two reactions. Moreover, phosphorylation of Thr233 appeared to be required for Pto–Pti1 physical interaction, as a mutation of this site to alanine, but not to aspartate, abolished the interaction between Pto and Pti1 in the yeast two‐hybrid system.
Assembly of the Rieske iron–sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts
Authors: Kapazoglou Aliki , Mould Ruth M. , Gray John C.
Published: March 2003

The assembly of the Rieske iron–sulphur protein into the cytochrome bf complex was examined following import of 35S‐labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside‐solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe‐2S] centre; the mutant Δ45–52 which removed a glycine‐rich region predicted to form a flexible hinge between the hydrophobic membrane‐associated region and the hydrophilic lumenal domain; and mutants Δ168–173 and Δ177–179 which removed two C‐terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe–2S] cluster, the glycine‐rich region and the C‐terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts.
A transglycosylating 1,3(4)‐β‐glucanase from Rhodothermus marinus
Authors: Petersen Bent O. , Krah Martin , Duus Jens Ø. , Thomsen Karl K.
Published: March 2003

The enzymatic hydrolysis of polysaccharides by the 1,3(4)‐β‐glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3‐β‐linkages of 3‐O‐substituted glucose units in 1,3‐β‐glucans such as laminarin and curdlan, and also the 1,4‐β‐linkages of 3‐O‐substituted β‐glucose in β‐glucans such as lichenin and 1,3‐1,4‐β‐glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley β‐glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one‐dimensional and two‐dimensional 1H‐NMR and 13C‐NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1,3‐β‐glycosidic and 1,4‐β glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4‐O‐glucosyl‐laminaribiose from exclusively 1,3‐β‐oligoglucosides. When barley 1,3‐1,4‐β‐glucan was incubated with LamR the β‐1,4‐linkages of 3‐O‐substituted β‐glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3‐β‐glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme–substrate complex and subsequent hydrolysis/transglycosylation.
Phylloxin, a novel peptide antibiotic of the dermaseptin family of antimicrobial/opioid peptide precursors
Authors: Pierre Thierry N. , Seon Aurélia A. , Amiche Mohamed , Nicolas Pierre
Published: March 2003

A novel family of peptide precursors that have very similar N‐terminal preprosequences followed by markedly different C‐terminal domains has been identified in the skin of hylid frogs belonging to the genus Phyllomedusinae. Biologically active peptides derived from the variable domains include the dermaseptins, 28–34‐residue peptides that have a broad‐spectrum microbicidal activity, and dermorphin and the deltorphins, d‐amino acid containing heptapeptides that are very potent agonists for the µ‐opioid and δ‐opioid receptors, respectively. This report describes the isolation, synthesis and cloning of phylloxin, a prototypical member of a novel family of antimicrobial peptides derived from the processing of a dermaseptin/dermorphin‐like precursor. The structure of phylloxin (GWMSKIASGIGTFLSGIQQ amide) shows no homology to the dermaseptins, but bears some resemblance to the levitide‐precursor fragment and the xenopsin‐precursor fragment, two antimicrobial peptides isolated from the skin of an evolutionarily distant frog species, Xenopus laevis. Circular dichroism spectra of phylloxin in low polarity medium, which mimics the lipophilicity of the membrane of target microorganisms, indicated 60–70% α‐helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning residues 1–19. Phylloxin is an addition to the structurally and functionally diverse peptide families encoded by the rapidly evolving C‐terminal domains of the dermorphin/dermaseptin group of precursors.
Cdc20 protein contains a destruction‐box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase‐promoting complex
Authors: Goh Phuay‐Yee , Lim Hong Hwa , Surana Uttam
Published: March 2003

Both chromosome segregation and the final exit from mitosis require a ubiquitin‐protein ligase called anaphase‐promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction‐box sequence in the N‐terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat‐containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC‐mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell‐cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction‐box containing proteins, such as Cdc20, in an APC‐independent manner.
Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter
Authors: Niedziela‐Majka Anita , Kochman Marian , Ożyhar Andrzej
Published: March 2003

The functional 20‐hydroxyecdysone (20E) receptor is a heterodimer of two members of the nuclear hormone receptors superfamily; the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. As most of the natural 20E‐response elements are highly degenerated palindromes, we were interested in determining whether or not such asymmetric elements could dictate the defined orientation of the Usp/EcR complex. We have investigated interaction of EcR and Usp DNA‐binding domains (EcRDBD and UspDBD, respectively) with the palindromic response element from the hsp27 gene promoter (hsp27pal). The hsp27pal half‐sites contribute differently to the binding of the heterodimer components; the 5′ half‐site exhibits higher affinity for both DBDs than the 3′ half‐site. This observation, along with data demonstrating that UspDBD exhibits approximate fourfold higher affinity to the 5′ half‐site than EcRDBD, suggest that UspDBD locates the EcRDBD/UspDBD heterocomplex in the defined orientation (5′‐UspDBD‐EcRDBD‐3′) on the hsp27pal sequence. The binding polarity onto hsp27pal is accompanied by different contribution of the UspDBD and EcRDBD C‐terminal sequences to the DNA‐binding and heterocomplex formation. This is supported by finding that deletion of the C‐terminal of EcRDBD region corresponding to the putative A‐helix severely decreased binding of the EcRDBD to the hsp27pal. In contrast, UspDBD in which corresponding residues were deleted exhibited the same hsp27pal binding pattern as the wild type UspDBD. Additional truncation comprising the putative T‐box, resulted in a reduced binding of the mutated UspDBD. This truncation however, still allowed effective EcRDBD/UspDBD heterodimer formation. Finally we demonstrated that perfect palindromes, composed of two hsp27pal 5′ half‐sites (or of the related sequence) contain all of the structural information necessary for the anisotropic UspDBD/EcRDBD heterocomplex formation. However, the perfect palindromes bind isolated homomeric DBDs as well as their heterocomplex with higher affinity than imperfect hsp27pal .

This is the first report indicating that natural 20E response elements, which with one exception are degenerated palindromes, may act as functionally asymmetric elements in a manner similar to the action of direct repeats in vertebrates.
The hybrid‐cluster protein (‘prismane protein’) from Escherichia coli
Authors: van den Berg Willy A. M. , Hagen Wilfred R. , van Dongen Walter M. A. M.
Published: March 2003

Hybrid‐cluster proteins (‘prismane proteins’) have previously been isolated and characterized from strictly anaerobic sulfate‐reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe–4S] cubane cluster with spin‐admixed S = 3/2 ground‐state paramagnetism and a novel type of hybrid [4Fe–2S–2O] cluster, which can attain four redox states.

Genomic sequencing reveals that genes encoding putative hybrid‐cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid‐cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid‐cluster protein, however, give evidence for the presence of a [2Fe–2S] cluster instead of a [4Fe–4S] cluster. The hcp gene encoding the hybrid‐cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe–2S] cluster as cofactors. It catalysed the reduction of the hybrid‐cluster protein with NADH as an electron donor. Midpoint potentials (25 °C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: −320 mV) are transferred along the [2Fe–2S] cluster of the NADH oxidoreductase (Em = –220 mV) and the [2Fe–2S] cluster of the hybrid‐cluster protein (Em = –35 mV) to the hybrid cluster (Em = –50, +85 and +365 mV for the three redox transitions).

The physiological function of the hybrid‐cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate‐and/or nitrite respiration.
Solution structure of oxidized microsomal rabbit cytochrome b5
Authors: Banci Lucia , Bertini Ivano , Rosato Antonio , Scacchieri Serena
Published: March 2003

Cytochrome b5 is heterogeneous in solution because of the presence of two isomers (A and B), differing in the rotation of the heme plane around the axis defined by the α and γ meso protons. For rabbit cytochrome b5, the A/B ratio is 5 : 1. The solution structure of the major form of the oxidized soluble fragment of rabbit microsomal cytochrome b5 (94 amino acids) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 3J couplings, a family of 40 energy‐minimized conformers were obtained with average backbone rmsd (for residues 4–84) of 0.060 ± 0.016 nm and average target function of 0.0078 nm2, no distance violations being larger than 0.03 nm. The structure was compared with the solution structures of the A (major) and B (minor) isomers of the rat cytochrome in the oxidized form. The A/B ratio for the rat cytochrome is 1.5 : 1, despite the very high sequence similarity (93%) to the rabbit protein. This comparison has provided insights into the factors determining the distribution in solution of the two isomers differing with respect to heme orientation. It appears that residues 23 and 74 are both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interactions are the key factors in determining the relative stability of one isomer with respect to the other.
The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA‐dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 °C
Authors: Spies Maria , Kil Yuri , Masui Ryoji , Kato Ryuichi , Kujo Chizu , Ohshima Toshihisa , Kuramitsu Seiki , Lanzov Vladislav
Published: March 2003

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 °C. It also showed high stability of 18.3 kcal·mol−1 (76.5 kJ·mol−1) at 25 °C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA‐like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA‐dependent manner and to catalyze DNA strand exchange. At 75 °C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 °C. The cooperativity of ATP hydrolysis and ssDNA‐binding ability of the protein above 75 °C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA‐dependent ATPase activity of P. islandicum RadA displays a temperature‐dependent capacity to exist in two different catalytic modes, with 75 °C being the critical threshold temperature.
Use of proteoliposomes to generate phage antibodies against native AMPA receptor
Authors: Jespersen Lene K. , Kuusinen Arja , Orellana Adelina , Keinänen Kari , Engberg Jan
Published: March 2003

To isolate antibodies against ionotropic glutamate receptors (GluRs), we prepared a phage antibody library from mice immunized with proteoliposomes containing purified α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), a selective GluRD receptor. Specific binders were selected by repeated rounds of affinity panning against immobilized GluRD liposomes. Using this approach, we obtained a panel of high‐affinity antibody fragments that immunoprecipitated both recombinant and native GluRD receptors, but not GluR6, a kainate receptor subunit with a 40% sequence similarity. The antibody fragments showed subunit selectivity, some being strictly specific for GluRD, whereas others also recognized the GluRB and GluRC but not GluRA subunits. Further experiments indicated that the epitopes recognized were conformational in nature and reside in the N‐terminal extracellular 400‐residue X domain of GluRD. Our results suggest that proteoliposomes, in combination with phage display technology, provide an effective tool for the generation of high‐affinity conformation‐sensitive monoclonal antibodies against predetermined membrane proteins.
Structural studies of the O‐specific polysaccharide from Plesiomonas shigelloides strain CNCTC 113/92
Authors: Czaja Jolanta , Jachymek Wojciech , Niedziela Tomasz , Lugowski Czeslaw , Aldova Eva , Kenne Lennart
Published: March 2003

The structure of the O‐specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix‐assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure:

in which d‐β‐d‐Hepp is dglycero‐β‐dmanno‐heptopyranose and 6d‐β‐d‐Hep is 6‐deoxy‐β‐dmanno‐heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O‐antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high‐resolution magic angle spinning technique, 1H‐NMR spectra were also obtained for the O‐polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.
Characterization of cis‐acting elements in the promoter of the mouse metallothionein‐3 gene
Authors: Faraonio Raffaella , Moffatt Pierre , LaRochelle Olivier , Schipper Hyman M. , S‐Arnaud René , Séguin Carl
Published: March 2003

The metallothionein (MT)3 gene is expressed predominantly in the brain and the organs of the reproductive system, and fails to respond to metal ions in vivo. A CTG repeat was proposed to function as a potential repressor element in nonpermissive cells, and a sequence similar to the JC virus silencer element was found to function as a negative element in permissive primary astrocytes. The objective of this study was to characterize further the mechanisms governing cell‐type specific MT‐3 gene transcription. We searched for a suitable cell line expressing the MT‐3 gene to be used for determination of MT‐3 promoter tissue specificity, and showed that MT‐3 expression is activated during neuroectodermal differentiation of P19 cells induced by retinoic acid to levels similar to those found in whole brain. Deletion of the CTG repeat or of the JC virus silencer did not promote MT‐3 promoter activity in nonpermissive cells, or enhance expression in permissive cells. We identified MT‐3 promoter sequences interacting with liver and brain nuclear proteins, as assayed by DNase I footprinting analyses and electrophoretic mobility shift assay, and assessed the role of these sequences in the regulation of MT‐3 expression by cotransfection experiments. We generated stable transfectants in permissive C6 and nonpermissive NIH‐3T3 cells, and analysed the methylation status of the MT‐3 gene. These studies show that regulation of tissue‐specific MT‐3 gene expression does not appear to involve a repressor, and suggest that other mechanisms such as chromatin organization and epigenetic modifications could account for the absence of MT‐3 gene transcription in nonpermissive cells.
Epidermal growth factor receptor is modulated by redox through multiple mechanisms
Authors: Kamata Hideaki , Shibukawa Yukinao , Oka Shin‐Ichi , Hirata Hajime
Published: March 2003

The cellular redox state has been shown to play an essential role in cellular signaling systems. Here we investigate the effects of reductants and H2O2 on the signaling of epidermal growth factor (EGF) in cells. H2O2 induced the phosphorylation of the EGF receptor and the formation of a receptor complex comprising Shc, Grb2, Sos, and the EGF receptor. Dimerization or oligomerization of the EGF receptor was not induced by H2O2. Protein tyrosine phosphatase (PTP) assay showed that H2O2 suppressed dephosphorylation of the EGF receptor in cell lysates, suggesting that inactivation of PTP was involved in H2O2‐induced activation of the EGF receptor. In contrast, the reductants N‐acetyl‐l‐cysteine [Cys(Ac)] and dithiothreitol markedly suppressed EGF‐induced dimerization and activation of the EGF receptor in cells. In accordance with suppression of the EGF receptor, Cys(Ac) suppressed EGF‐induced activation of Ras, phosphatidylinositol 3‐kinase and mitogen‐activated protein kinase. Dithiothreitol completely inhibited EGF binding and kinase activation of the EGF receptor both in vitro and in vivo. In contrast, Cys(Ac) suppressed high‐affinity EGF‐binding sites on the cells, but had no effect on low‐affinity binding sites. Furthermore, Cys(Ac) did not suppress EGF‐induced kinase activation or dimerization of the EGF receptor in vitro, indicating that it suppressed the EGF receptor through a redox‐sensitive cellular process or processes. Thus, the EGF receptor is regulated by redox through multiple steps including dephosphorylation by PTP, ligand binding, and a Cys(Ac)‐sensitive cellular process or processes.