bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of riboflavin in Archaea
Authors: Haase Ilka , Mörtl Simone , Köhler Peter , Bacher Adelbert , Fischer Markus
Published: March 2003

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7‐dimethyl‐8‐ribityllumazine, by condensation of 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione and 3,4‐dihydroxy‐2‐butanone 4‐phosphate. Steady state kinetic analysis at 37 °C and pH 7.0 indicated a catalytic rate of 11 nmol·mg−1·min−1; Km values for 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione and 3,4‐dihydroxybutanone 4‐phosphate were 12.5 and 52 µm, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative‐stained electron micrographs showed predominantly spherical particles with a diameter of about 150 Å. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.
Biosynthesis of the topaquinone cofactor in copper amine oxidases
Authors: Rinaldi Andrea C. , Porcu Cristina M. , Oliva Stefania , Curreli Nicoletta , Rescigno Antonio , Sollai Francesca , Rinaldi Augusto , Finazzi‐Agró Alessandro , Sanjust Enrico
Published: March 2003

Copper amine oxidases utilize 2,4,5‐trihydroxyphenylalanine quinone (topaquinone) as a cofactor in enzymatic catalysis. This cofactor is formed from a tyrosine residue through a self‐catalytic mechanism with the participation of the copper ion at the active site. Although pathways have been postulated for topaquinone biogenesis, portions of this scheme are still unclear. We utilized 4‐tert‐butyl‐derived models for the putative intermediates of topaquinone generation and studied the effect of Cu(II) and Zn(II) ions on each autoxidative step from dopa‐ to topaquinone‐like compounds at physiological pH (7.4). Several polyvinyl‐alcohol‐based soluble resins bearing mono‐ and di‐hydroxyphenolic moieties were also prepared, and their tendency to give hydroxyquinonic structures when incubated at alkaline pH values was investigated. Our results confirm (although indirectly) the formation of dopa and dopaquinone during topaquinone biosynthesis. Moreover, we collected evidence that, following the formation of dopa, the role of the active‐site copper ion in topaquinone biogenesis would be limited to the catalysis of the two subsequent quinonization steps (i.e. from dopa to dopaquinone and from topa to topaquinone), thus disfavoring the possibility of a direct intervention of the metal ion in the hydroxylation of dopaquinone. In particular, Cu(II) was shown to influence deeply the autoxidation of 1,2,5‐trihydroxy‐4‐tert‐butylbenzene, used as model of topa, both increasing the reaction rate and changing its mechanism. The mechanistic implications of these findings for the biogenesis of topaquinone and its analogs at the active site of various amine oxidases are discussed.
Role of Arg180 of the D2 protein in photosystem II structure and function
Authors: Manna Pradip , LoBrutto Russell , Eijckelhoff Camiel , Dekker Jan P. , Vermaas Wim
Published: March 2003

On the basis of sequence comparison with the M subunit of the reaction center of purple bacteria, no residues in photosystem II can be clearly identified that may be predicted to correspond to the His residue that binds one of the accessory bacteriochlorophylls in the purple bacterial reaction center. However, the Arg180 residue of the D2 protein is close to where this residue is predicted to be and could conceivably serve as a chlorophyll ligand. To analyze the function of Arg180, it was changed to nine different amino acids in the cyanobacterium Synechocystis sp. PCC 6803. Except for the Arg180←Gln (R180Q) mutant, the resulting strains were no longer photoautotrophic. The properties of photosystem II upon mutation of Arg180 were probed in strains from which photosystem I had been deleted genetically. Mutations at the Arg180 residue affected oxygen evolution capacity and the amount of photosystem II that was present in thylakoids. Surprisingly, in the Arg180 mutants, EPR signals that may originate from the oxidized redox‐active Tyr160 of the D2 protein (Y.esox.rb.eiD.rb) were small and generally did not resemble the usual signal IIs, signifying an effect of the Arg180 mutations on the environment surrounding Tyr160. In addition, in most mutants, the charge recombination kinetics between the primary electron‐accepting quinone in photosystem II (Q.es−.rb.eiA.rb) and oxidized species on the donor side were faster upon introducing mutations at Arg180 suggesting an increased steady‐state concentration of P680+ in the mutants. However, Arg180 mutations also affected Q.es−.rb.eiA.rb oxidation by the secondary electron‐accepting quinone (QB). HPLC analysis showed that, in the Arg180 mutants that were assayed, the pheophytin/chlorophyll ratio of photosystem II had not changed, indicating that the mutations did not lead to a pheophytinization of one of the chlorophyll molecules. Even though the results presented do not provide positive evidence that Arg180 of the D2 protein corresponds in function to the ligand to the central Mg in an accessory bacteriochlorophyll in reaction centers of purple bacteria, it is clear that changes in Arg180 greatly affect Tyr160 and P680. Various scenarios are discussed that are compatible with the data presented, and include an apparently close interaction between Arg180, His189, and Tyr160, and the possibility of the involvement of multiple chlorophylls to together form P680.
Site‐specific modification of 4.5S RNA apical domain by complementary oligodeoxynucleotides carrying an alkylating group
Authors: Bulygin Konstantin , Malygin Alexey , Karpova Galina , Westermann Peter
Published: March 2003

Site‐specific alkylation of RNA by reactive oligodeoxynucleotides provides structural information and represents the first step towards the design of RNA derivatives to be used for functional studies. Specific alkylation of 4.5S RNA at G53, the first base of the apical tetraloop, was achieved by incubation with oligodeoxynucleotide ON2, complementary to nucleotides 38−53, which carries a p‐(N‐2‐chloroethyl‐N‐methylamino)benzylamidophosphate group at the 5′ end. Alkylation efficiency was increased by a factor of 6, without alteration of specificity, in the presence of a helper oligodeoxynucleotide, ON1, complementary to nucleotides 58−71 of the opposite strand of the RNA helix. A second reactive oligodeoxynucleotide, ON1‐3′‐R, was obtained by attaching the alkylating group to the 3′ end of ON1. ON1‐3′‐R was able to modify G58. In the presence of ON2 as a helper oligodeoxynucleotide, the specificity of ON1‐3′‐R changes and efficient alkylation of nucleotides G54, A56 and G57 of the apical region of 4.5S RNA was observed.
The PC motif : a novel and evolutionarily conserved sequence involved in interaction between p40 phox and p67phox, SH3 domain‐containing cytosolic factors of the phagocyte NADPH oxidase
Authors: Nakamura Rika , Sumimoto Hideki , Mizuki Kazuhito , Hata Kenichiro , Ago Tetsuro , Kitajima Shigetaka , Takeshige Koichiro , Sakaki Yoshiyuki , Ito Takashi
Published: March 2003

The superoxide‐generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane‐integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40 phox. While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67phox using the yeast two‐hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C‐terminal region of p40 phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C‐terminal region of p40 phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal‐transducing proteins from yeast to human. Intensive site‐directed mutagenesis to the motif in p40 phox revealed that it plays a critical role in the binding to p67 phox. Thus the PC motif appears to represent a novel module for protein−protein interaction used in a variety of signaling pathways.
Immunochemical and functional characterization of an agonist‐like monoclonal antibody against the M2 acetylcholine receptor
Authors: Elies Rozenn , Fu L. X. Michael , Eftekhari Pierre , Wallukat Gerd , Schulze Wolfgang , Granier Claude , Hjalmarson Åke , Hoebeke Johan
Published: March 2003

Monoclonal antibodies were raised against a peptide corresponding to the second extracellular loop of the M2 acetylcholine receptor. One of the monoclonal antibodies, B8E5, was selected for further characterization on the basis of its high yield, its isotype (IgG2a), its dissociation kinetics and its agonist‐like activity.

The epitope recognized by B8E5 corresponded to the N‐terminal part of the second extracellular loop of the receptor (V‐R‐T‐V‐E‐) as determined by competition immunoassays and epitope scanning. The KA of B8E5 for the target peptide was assessed by surface plasmon resonance (SPR) to be 6.5×107 M−1 by equilibrium and 3.7×107 M−1 by kinetic analysis. B8E5 recognized the M2 acetylcholine receptor on rat cardiac tissue. It only recognized the non‐reduced receptor in immunoblots. The antibody had no effect on antagonist binding but decreased the affinity for the agonist carbachol.

B8E5 decreased the beating frequency of neonatal rat cardiomyocytes. The effect was specific since it was blocked by the target peptide and the antagonist atropine. The EC50 of the antibody corresponded to the KA measured by surface plasmon resonance. The physiological effect of the antibody did not lead to desensitization. The Fab fragments had no physiological effect; subsequent addition of anti‐mouse IgG however restored the physiological effect.

These results confirm that the N‐terminus of the second extracellular loop is a functional target for antibodies against the M2 acetylcholine receptor. They suggest that the functional epitope is only accessible in the non‐reduced receptor. The antibodies act through a functional dimerization of the receptor.
Stop‐transfer function of pseudo‐random amino acid segments during translocation across prokaryotic and eukaryotic membranes
Authors: Sääf Annika , Wallin Erik , von Heijne Gunnar
Published: March 2003

We have measured the efficiency of stop‐transfer function for a set of pseudo‐random, 18‐residue amino acid segments, both in Escherichia coli and in mammalian microsomes. In general, stop‐transfer function correlates well with the mean hydrophobicity of the segment, though exceptions exist. Kinetic studies suggest that polar segments are rapidly translocated through the E. coli inner membrane and that strongly hydrophobic segments become permanently anchored, while sequences with an intermediate mean hydrophobicity become partly trapped in a transmembrane disposition for a considerable time before being released to the periplasm or degraded.
Modes of action on cotton and bacterial cellulose of a homologous endoglucanase‐exoglucanase pair from Trichoderma reesei
Authors: Srisodsuk Malee , Kleman‐Leyer Karen , Keränen Sirkka , Kirk T. Kent , Teeri Tuula T.
Published: March 2003

The endoglucanase I (EGI) and the cellobiohydrolase I (CBHI) of the filamentous fungus Trichoderma reesei form a homologous pair of cellulolytic enzymes which nevertheless have different modes of action. We show here that the action of CBHI on bacterial microcrystalline cellulose results in efficient solubilisation but only a slow decrease in its degree of polymerisation. In contrast, the action of EGI results in a rapid decrease of the degree of polymerisation but less efficient overall solubilisation of the substrate. CBHI alone was practically inactive toward cotton which has a high initial degree of polymerisation and a complex morphology. EGI rapidly reduced the degree of polymerisation of cotton, and slowly solubilised part of it. Working synergistically, EGI and CBHI solubilised cotton more rapidly and to a greater extent than EGI alone. Our data are consistent with the exoglucanase nature of CBHI and also provide some evidence supporting its processive mode of action.
Sorting of invertase signal peptide mutants in yeast dependent and independent on the signal‐recognition particle
Authors: Rothe Christine , Lehle Ludwig
Published: March 2003

There is growing evidence that yeast contains two efficient pathways of protein translocation across the endoplasmic reticulum membrane, one dependent on the signal‐recognition particle (SRP) and the other independent. Their specificity, however, is largely obscure. For higher eukaryotes it has been shown that a high average hydrophobicity of the core region with a minimal length around six or seven amino acids, as well as a stabilized α‐helix, are decisive structural features for translocation. Using yeast invertase as a secretory model protein, we have found that mutated signal sequences with Pro or Gly in the core, or having only four hydrophobic amino acids, are not functional in translocation across microsomal membranes of dog pancreas because they do not interact with the SRP. Expression of these mutant variants in Saccharomyces cerevisiae revealed that they are sorted independently of the SRP since translocation was not impaired in an SRP‐deficient yeast strain. In contrast to this, wild‐type invertase is translocated SRP‐dependently in wild‐type cells and shows a decreased translocation in SRP‐deficient cells.

By overexpression of Srp54p, but not of Hsc70p, the translocation defect of wild‐type invertase in an SRP54 disruptant is restored. The data indicate that targeting of proteins to the endoplasmic reticulum in Saccharomyces cerevisiae seems to be more flexible than in higher eukaryotes as far as the structural requirements of signal sequences are concerned, and that the route taken is specified by the sequence.
Probing the mechanism of an Mn2+‐dependent ribozyme by means of platinum complexes
Authors: Bombard Sophie , Kozelka Jiří , Favre Alain , Chottard Jean‐Claude
Published: March 2003

The smallest ribozyme system known is the pentanucleotide GAAACp, which is specifically cleaved by Mn2+, in the presence of poly(U), generating guanosine 2′,3′‐cyclic phosphate and AAACp. A plausible mechanism has been proposed, involving the participation of two Mn2+ with structural and catalytic roles, the first one cross‐linking the two N7 atoms of G1 and A4, and the other binding to the N7 atom of A2 and activating the 2′‐OH group of G1 [Kazakov, S. & Altman, S. (1992) Proc. Natl Acad. Sci. USA 89, 7939−7943]. In the present work, we have utilized the high affinity of Pt(II) complexes for N7 atoms of purines in an attempt to form a stable active ribozyme by replacing the structural Mn2+ by Pt2+. We thus replaced the proposed kinetically labile G1N7‐Mn2+‐A4N7 cross‐link by an inert N7‐trans‐Pt(NH3).es2+.rb.ei2.rb‐N7 cross‐link. In a complementary investigation, the N7 atoms of the individual purines of GAAACp were selectively blocked by a Pt(NH3).es2+.rb.ei3.rb residue to determine which of the N7 sites participate in the Mn2+‐mediated cleavage. Other N7‐PtII‐N7 crosslinks were also investigated. Accordingly, we have prepared four monoadducts, each bearing the Pt(NH3).es2+.rb.ei3.rb residue on one of the purines and a series of chelates of trans‐Pt(NH3).es2+.rb.ei2.rb and cis‐Pt(NH3).es2+.rb.ei2.rb and have tested them for Mn2+‐induced cleavage. Binding of Pt(NH3).es2+.rb.ei3.rb to G1 or A4 did not alter the efficiency of the specific cleavage between G1 and A2 catalyzed by Mn2+/poly(U), whereas cross‐linking of G1 and A4 by trans‐Pt(NH3).es2+.rb.ei2.rb inhibited it completely. Hence, a cross‐link between G1 and A4 is not required for the site‐specific cleavage. Binding of Pt(NH3).es2+.rb.ei3.rb to A2 or A3 strongly inhibits the G1/A2 cleavage, suggesting that these bases are likely to be involved in manganese coordination in the cleaving complex. A site‐specific Mn2+‐dependent cleavage between A4 and C5 was observed for the G1‐A4 and G1‐A3 adducts cross‐linked by trans‐Pt(NH3).es2+.rb.ei2.rb, the G1‐A2 adduct cross‐linked by cis‐Pt(NH3).es2+.rb.ei2.rb, and the three monoadducts bearing the Pt(NH3).es2+.rb.ei3.rb residue on G1, A2 or A3; poly(U) did not exert any influence on this cleavage.
Fibrin selectivity of the isolated protease domains of tissue‐type and vampire bat salivary gland plasminogen activators
Authors: Toschi Luisella , Bringmann Peter , Petri Thomas , Donner Peter , Schleuning Wolf‐Dieter
Published: March 2003

The activity of vampire bat (Desmodus rotundus) salivary plasminogen activator (D. rotundus PAα1) and to a much lesser extent of tissue‐type plasminogen activator (t‐PA) is stimulated by the presence of fibrin. This cofactor requirement has in the past intuitively been attributed to fibrin binding. We have previously shown that elements of the non‐protease domain of D. rotundus PAα1 could contribute to fibrin stimulation irrespective of fibrin binding.

We now demonstrate that the protease domain of D. rotundus PAα1 by itself exhibits fibrin selectivity, i.e. it is 32‐fold stimulated by fibrin but only 1.5‐fold by fibrinogen. To a lesser extent this fibrin selectivity is also shared by the protease domain of t‐PA. Our findings indicate that protein‐protein interactions apart from fibrin binding affect the stimulatory mechanism of fibrin on D. rotundus PAα1 and t‐PA.
Human cancer cell lines growth inhibition by GTn oligodeoxyribonucleotides recognizing single‐stranded DNA‐binding proteins
Authors: Scaggiante Bruna , Morassutti Carla , Dapas Barbara , Tolazzi Giuseppe , Ustulin Franca , Quadrifoglio Franco
Published: March 2003

Oligonucleotides can specifically target not only nucleic acids but also proteins. Some proteins recognizing oligonucleotides in a sequence‐specific manner have been related to cancer transformation and progression. We have found that oligonucleotides composed by repeated and/or variable intervals of GTn with 1  n  7, are able to exert a specific and dose‐dependent growth inhibition on human CCRF‐CEM, CEM‐VLB300, U937, Jurkat, H9 and HeLa tumor cell lines. In contrast, G←C, G←A, T←C and T←A base substituted control oligonucleotides do not significantly alter cellular growth. In all cell lines, a nuclear protein (molecular mass = 45 ± 7 kDa), which specifically recognizes GTn, was identified. Our hypothesis is that the formation of the GTn‐protein complex in human cancer cell lines may be involved in the growth inhibition effect. In fact, we found that the reduction or lack of cytotoxic effects by GTn in phorbol 12‐myristate 13‐acetate‐treated CCRF‐CEM cells and in normal human lymphocytes is paralleled by the simultaneous reduction or lack of GTn‐protein complex. Oligonucleotides specifically ’quenching' intracellular protein activities by forming oligonucleotide‐protein complexes may be of potential interest in the treatment of human tumors.
The V3 loop of human immunodeficiency virus type‐1 envelope protein is a high‐affinity ligand for immunophilins present in human blood
Authors: Endrich Michael M. , Gehring Heinz
Published: March 2003

Human immunodeficiency virus type‐1 (HIV‐1) infection requires binding of the envelope protein gp120 to host CD4 receptors and the action of the chemokine receptors CXCR4 or CCR5, which define cell tropism. The proline‐containing V3 loop of gp120 determines the selection of the chemokine receptor and participates in conformational changes on binding of gp120 to CD4. In this study, we show that macrophage‐tropic and T‐cell‐tropic V3 loop peptides bind specifically to the active site of the immunophilins FK506‐binding protein (FKBP12), and cyclophilins A and B. Macrophage‐tropic and T‐cell‐tropic V3 loop peptides inhibited the peptidyl‐prolyl cis‐trans isomerase (PPIase) activities of the immunophilins. Kd values in the range 0.036−4.1 μM were determined with V3 loop peptides labeled with an environmentally sensitive fluorophore. The observed binding properties of the V3 loop peptides reveal structural motifs of linear water‐soluble peptidic substrates for tight interaction with immunophilins. FKBP12, and cyclophilins A and B were found to be present in normal human blood in the ranges 0.8−1.7, 1.4−2.3 and 2.4−3.1 nM, respectively, as demonstrated by PPIase activity measurements and western blot analysis. Cyclophilins A and B levels in serum of HIV‐1−infected individuals were increased 3.6fold and 1.6‐fold. Due to the interaction of immunophilins with V3 loop peptides and with the envelope protein gp120, a role of immunophilins in HIV pathogenesis as conformases or docking mediators seems possible, since immunophilin receptors on cell membranes and immunophilin‐related virulence factors of pathogens have been identified.
Sequential phosphorylation of Tau by glycogen synthase kinase‐3β and protein kinase A at Thr212 and Ser214 generates the Alzheimer‐specific epitope of antibody AT100 and requires a paired‐helical‐filament‐like conformation
Authors: Zheng‐Fischhöfer Qingyi , Biernat Jacek , Mandelkow Eva‐Maria , Illenberger Susanne , Godemann Robert , Mandelkow Eckhard
Published: March 2003

AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Here we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), next of Thr212 by glycogen synthase kinase (GSK)‐3β (a proline‐directed kinase), then of Ser214 by protein kinase A (PKA). Conversely, if Ser214 is phosphorylated first it protects Thr212 and the Ser‐Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformation of tau induced by polyanions such as heparin, RNA or poly(Glu), conditions which also favor the formation of paired helical filaments. The Alzheimer‐like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK‐3β and PKA, which can be inhibited by their specific inhibitors LiCl and RII, respectively. A cellular model displaying the reaction with AT100 is presented by Sf9 insect cells transfected with Tau. Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.
Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen
Authors: Matsui Taei , Sakurai Yoshihiko , Fujimura Yoshihiro , Hayashi Izumi , Oh‐Ishi Sachiko , Suzuki Masami , Hamako Jiharu , Yamamoto Yoshinobu , Yamazaki Junko , Kinoshita Michiko , Titani Koiti
Published: March 2003

We have isolated a serine protease, halystase, from Agkistrodon halys blomhoffii venom by chromatography on DEAE‐Sepharose, heparin‐Sepharose and Q‐Sepharose columns, and have determined the complete amino acid sequence by Edman degradation and by mass spectral analysis of peptides generated by enzymatic and chemical cleavage. The 238‐residue sequence of halystase, containing N‐linked carbohydrates (about 13 %) at two sites showed significant similarity to other thrombin‐like snake venom serine proteases (66−72 %), mammalian tissue kallikrein (42 %) and thrombin (26 %). Halystase contained the tentative catalytic triad of His43, Asp88 and Ser184 common to all serine proteases and Asp178 in the primary substrate‐binding site. Although halystase contained an RGD sequence at residues 181−183, it did not inhibit platelet aggregation induced by ADP or collagen. It hydrolyzed most efficiently a tissue‐kallikrein substrate, prolylphenylalanylarginyl‐4‐methyl‐coumaryl‐7‐amide, and released bradykinin from bovine kininogen. Halystase did not coagulate human plasma, but it cleaved the fibrinogen B β chain at the carboxyl side of Arg42 and cleaved slowly the fibrogen A α chain. Fibrinogen thus treated gradually became insensitive to thrombin. The proteolytic activity was inhibited with diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride or leupeptin. These results indicate that halystase is a serine protease structurally similar to coagulating thrombin‐like snake venom proteases, but it specifically cleaves fibrinogen at sites different from thrombin without inducing fibrin clotting, and hydrolyzes kininogen to produce bradykinin, resulting in the reduction of blood pressure.
Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose
Authors: Cha Hyun‐Ju , Yoon Hyun‐Geun , Kim Young‐Wan , Lee Hee‐Seob , Kim Jung‐Wan , Kweon Ki‐Sung , Oh Byung‐Ha , Park Kwan‐Hwa
Published: March 2003

A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69 627‐Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47−70 % identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) I‐5 from an alkalophilic Bacillus I‐5 strain. In addition to starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10 % glucose, isoacarbose, containing an α‐1,6‐glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The β‐cyclodextrin−hydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site‐directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.
The function of the periplasmic Sud protein in polysulfide respiration of Wolinella succinogenes
Authors: Klimmek Oliver , Kreis Vera , Klein Carola , Simon Jörg , Wittershagen Axel , Kröger Achim
Published: March 2003

The periplasmic Sud protein was previously isolated as a sulfide dehydrogenase from Wolinella succinogenes. Sud modified by a C‐terminal His‐tag (Sud‐His6) was produced in Escherichia coli by expression of the sud gene. Sud‐His6 catalyzed thiocyanate formation from cyanide and polysulfide. The Vmax of this activity was more than one order of magnitude higher than that of sulfide oxidation by dimethylnaphthoquinone and that of polysulfide reduction by BH.es−.rb.ei4.rb. The apparent Km was less than 20 μM polysulfide. Polysulfide and not elemental sulfur was found to be the product of sulfide oxidation by dimethylnaphthoquinone, in contrast to the earlier view [Kreis‐Kleinschmidt, V., Fahrenholz, F., Kojro, E. & Kröger, A. (1995) Arch. Microbiol. 165, 65−68]. Sud‐His6 did not contain metal ions or other prosthetic groups. Replacement by site‐directed mutagenesis of the single cysteine residue of the Sud monomer caused complete loss of activity, while the exchange of the single histidine residue or of the lysine residue situated next to cysteine did not affect activity. In equilibrium dialysis, the Sud‐His6 monomer bound up to ten polysulfide sulfur atoms with a dissociation constant of 0.2 mM. Sud‐His6 loaded with polysulfide sulfur showed an absorption spectrum in the range of 350−400 nm; this spectrum differed from that of free polysulfide. Electron transport from H2 to polysulfide catalyzed by the membrane fraction of W. succinogenes was stimulated by the presence of small amounts of Sud‐His6. The apparent Km for polysulfide decreased sevenfold in the presence of saturating amounts of Sud‐His6 (1 μM Sud‐His6 dimer). Similar results were obtained with intact W. succinogenes cells containing low and high amounts of Sud. Sud appears to function as a polysulfide binding protein and probably binds polysulfide sulfur to its cysteine residue and transfers it to the substrate site of the membraneous polysulfide reductase.
Isolation and characterization of an evolutionary precursor of human monoamine oxidases A and B
Authors: Sablin Sergey O. , Yankovskaya Victoria , Bernard Suzanne , Cronin Ciarán N. , Singer Thomas P.
Published: March 2003

An interesting flavoprotein‐type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned [Schilling, B. & Lerch, K. (1995 a) Biochim. Biophys. Acta 1243, 529−537; Schilling, B. & Lerch, K. (1995 b) Mol. Gen. Genet. 247, 430−438]. The properties of this MAO, as well as a substantial part of its amino acid sequence, resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes. It differs from MAO A and B in several respects, however, including the fact that it is soluble and of peroxisomal location and that the FAD is non‐covalently attached.

We have overexpressed the fungal enzyme (MAO‐N) in Escherichia coli and isolated it in pure form. Since several of the observations of previous workers on MAO‐N could not be reproduced, we have reexamined its substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties. MAO‐N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme. Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots. Contrary to a previous report [Schilling, B. & Lerch, K. (1995 a) Biochim. Biophys. Acta 1243, 529−537], however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines. MAO‐N is irreversibly inhibited by stoichiometric amounts of both (−)deprenyl and clorgyline in a mechanism‐based reaction, forming flavocyanine adducts with N5 of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting a closer resemblance to MAO A than B. The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO‐N and comparison with similar values for MAO A and B again pointed to a greater similarity to the former than the latter.
Structural characterization of the lipopolysaccharide O‐antigen and capsular polysaccharide of Vibrio ordalii serotype O : 2
Authors: Sadovskaya Irina , Brisson Jean‐Robert , Khieu Nam Huan , Mutharia Lucy M. , Altman Eleonora
Published: March 2003

Structures of the capsular and O‐chain polysaccharides of Vibrio ordalii serotype O : 2, the causative agent of vibriosis in salmonid fish, were determined by high‐field NMR techniques, mass spectrometric methods and partial hydrolysis. Both polymers were shown to be composed of linear tetrasaccharide repeating units, having the structure :
[←4)‐β‐
D‐Glcp2NAc3NAN‐(1←4)‐β‐Glcp2NAc3NAmA‐(1←4)‐α‐
L‐Galp2NAc3NAcA‐(1←3)‐β‐
D‐Sugp‐(1←]n,
∣
Fo‐
L‐Ala
where Am = acetamidino; Fo = formyl; AN = amide, and Sug = 2‐acetamido‐2,6‐dideoxy‐
D‐xylo‐hexos‐4‐ulose.
Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP‐N‐acetylglucosamine enolpyruvyltransferase (MurA)
Authors: Schönbrunn Ernst , Svergun Dmitri I. , Amrhein Nikolaus , Koch Michel H. J.
Published: March 2003

The enzyme UDP‐N‐acetylglucosamine (UDP‐GlcNAc) enolpyruvyltransferase (MurA), the target of the antibiotic fosfomycin, was investigated by small‐angle X‐ray scattering (SAXS) and fluorescence spectroscopy to detect conformational changes that had been proposed on the basis of the crystal structure of unliganded and liganded MurA. The SAXS data indicate that binding of UDP‐GlcNAc to free enzyme results in substantial conformational changes, which can be interpreted as the transition from an open to a closed form. Fosfomycin did not affect the structure of free enzyme or sugar‐nucleotide‐bound MurA. Phosphoenolpyruvate (pyruvate‐P) appeared to induce a structural change upon addition to free enzyme, which differed from that observed upon binding of UDP‐GlcNAc. Fluorescence experiments were performed using the hydrophobic fluorescence probe 8‐anilino‐1‐naphthalene sulfonate (ANS). The fluorescence quenching of MurA/ANS solutions upon addition of UDP‐GlcNAc or pyruvate‐P was concentration dependent in a saturating manner, yielding apparent dissociation constants of Kd(UDP‐GlcNAc) = 59 μM and Kd(pyruvate‐P) = 240 μM. The results suggest that binding of substrates does not exclusively follow an ordered mechanism with UDP‐GlcNAc binding first, although binding of UDP‐GlcNAc to free enzyme is preferred and possibly influenced by pyruvate‐P. The reaction thus appears to follow an induced‐fit mechanism, in which the binding site for fosfomycin, and presumably also for pyruvate‐P, is created by the interaction of free enzyme with the sugar nucleotide. The methods described here provide a tool for the characterization of site‐directed mutants of MurA and the interaction of this enzyme with potential inhibitors.
Downstream targets of urokinase‐type plasminogen‐activator‐mediated signal transduction
Authors: Konakova Marina , Hucho Ferdinand , Schleuning Wolf‐Dieter
Published: March 2003

We have investigated cellular signalling events induced by urokinase‐type plasminogen activator (uPA) independent of its proteolytic activity. Treatment of the human fibrosarcoma cell line HT 1080 with diisopropylphosphorofluoridate‐inactivated uPA (Dip‐F−uPA) triggers a cascade of intracellular signals which are mediated by the specific cell surface receptor for uPA (uPAR). We have found that anti‐uPAR Ig precipitate the src‐type protein tyrosine kinases fyn, hck and lck, which belong to a family of structurally and functionally related effectors participating in signalling from antigen and cytokine receptors. Of the three uPAR‐associated kinases, only hck is activated by uPA, whereas no changes in the activities of either fyn or lck could be detected by an in vitro immune complex kinase assay. We identified p38 and extracellular‐signal‐regulated kinase 2 from the mitogen‐activated protein kinase family as downstream components of a set of consecutive signalling molecules which teleologically alter the program of gene expression. Exposure of cells to uPA results in a significant increase in c‐fos mRNA that is partially due to an elevated rate of gene transcription. Presumably, the activation of the c‐fos gene leads to the subsequent formation of the transcription factor activator protein‐1 (AP‐1), since accumulation of c‐fos mRNA is followed by induction of target genes sensitive to AP‐1 such as plasminogen activator inhibitor type 2 (PAI‐2). These results provide new insights into proteolysis‐independent cytokine‐like effects of uPA.
The reductase RedA2 of the multi‐component dioxin dioxygenase system of Sphingomonas sp. RW1 is related to class‐I cytochrome P450‐type reductases
Authors: Armengaud Jean , Timmis Kenneth N.
Published: March 2003

The first step in the oxidation of the diaryl ethers dibenzo‐p‐dioxin and dibenzofuran by the bacterium Sphingomonas sp. RW1 is carried out by an atypical multi‐component ring hydroxylating dioxygenase. This heteromeric enzyme requires the participation of a flavoprotein, reductase A2, and an iron‐sulfur protein, Fdx1, to mediate the transfer of electrons from NADH to the dioxygenase for oxygen activation [Bünz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467−6475]. From the type of ferredoxin (Fd) and flavoprotein, this complex is presumed to belong to the class‐IIA dioxygenase system which has not been genetically analysed so far. The gene encoding the flavoprotein was identified by screening a genomic library constructed in pLAFR3 with a probe generated by PCR amplification. The nucleotide sequence of a 2.0‐kb DNA fragment encompassing the reductase gene, redA2, was determined. The specified protein shares 37−40 % identity with class‐I cytochrome P450 reductases and 27−35 % identity with reductases acting with class‐IIB dioxygenases. An FAD‐binding amino acid consensus sequence, as well as an NADH‐binding site were detected by analogy beginning at residues 10 and 153, respectively. The redA2 gene is not linked to the dioxin dioxygenase cistrons. The rare start codon, GTG, of the reductase was changed to ATG and the modified gene hyperexpressed in Escherichia coli using the strong T7 polymerase promoter. The recombinant reductase was purified to homogeneity with an approximate yield of 3.3 mg/g wet mass cells. Flavin analysis confirmed the presence of 1 FAD/mol protein. The Km values for NADH and Fdx1 are 22 ( ± 3) μM and 3.7 ( ± 0.4) μM, respectively.
Rules for the addition of O‐linked N‐acetylglucosamine to secreted proteins in Dictyostelium discoideum
Authors: Jung Eva , Gooley Andrew A. , Packer Nicolle H. , Karuso Peter , Williams Keith L.
Published: March 2003

One class of O‐glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N‐acetylglucosamine residue to Ser and Thr residues on secreted or membrane‐bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O‐glycosylation was used to further characterise the specificity of the UDP‐GlcNAc :polypeptide N‐acetylglucosaminyltransferase(s). Glutathione S‐transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2‐like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray‐ionisation mass spectrometry and protein sequencing showed that the modification is a single N‐acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in α configuration as determined by NMR studies.
Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state
Authors: Arteel Gavin E. , Thurman Ronald G. , Raleigh James A.
Published: March 2003

2‐Nitroimidazoles, such as pimonidazole, are reduced in cells with low oxygen tension and are, therefore, used as hypoxia markers. However, the effect of the pyridine nucleotide redox state on pimonidazole reduction is not known. Therefore, livers from fed or fasted rats were perfused with oxygen‐saturated buffer containing pimonidazole (400 μM) in the presence and absence of an inhibitor of the mitochondrial respiratory chain, potassium cyanide; these treatments were used to modulate the mitochondrial and cytosolic pyridine nucleotide redox states. Pimonidazole‐induced increases in oxygen uptake over basal values were as follows : fed, 15.1 ± 2.4; fasted, 4.2 ± 0.8; fed + KCN, 32.1 ± 0.9; fasted+KCN, 0.2 ± 0.2 μmol g−1 h−1. However, if NADPH was added in excess, microsomal oxygen uptake due to oxidative metabolism of pimonidazole was independent of treatment. These results indicate that pimonidazole‐stimulated O2 uptake, due predominantly to N‐oxidation and glucuronidation, is dependent on the NADPH redox state. In contrast, reduced pimonidazole adducts, detected immunochemically, accumulated in pericentral regions in liver. Increasing the NADH redox state by inhibiting the mitochondrial respiratory chain with KCN decreased protein‐bound pimonidazole adducts. Concomitantly, the average O2 tension of the liver was increased at least 30 %. However, KCN had no effect on total pimonidazole adducts detected by ELISA, although both cytosolic (lactate/pyruvate) and mitochondrial (3‐hydroxybutyrate/acetoacetate) NADH redox states were elevated by at least a factor of eight. These results indicate that, unlike oxidative metabolism, the pyridine nucleotide redox state does not determine the rate of reductive metabolism of pimonidazole. Instead, the cellular oxygen tension regulates this process. Therefore, even in cases where the supply of reducing equivalents is increased (e.g., ethanol metabolism), accumulation of the reduced bound product of pimonidazole is oxygen dependent in liver.
Photophysical properties of porphyrin planar aggregates in liposomes
Authors: Ricchelli Fernanda , Gobbo Silvano , Moreno Giuliana , Salet Christian , Brancaleon Lorenzo , Mazzini Alberto
Published: March 2003

This paper describes studies of some photophysical properties of non‐covalent planar aggregates of hematoporphyrin and protoporphyrin. This porphyrin species has been recently discovered and can be generated in lipid bilayers such as liposomes and inner mitochondrial membranes. The relative weight of this species in different media, as compared to porphyrin monomers and stacked aggregates, has been deduced by fluorescence decay studies. In contrast with what is observed for stacked aggregates, promotion of planar suprastructures can occur both in aqueous and lipid environments. The spectroscopic properties are very similar to those of the corresponding monomers, in particular as regards the shape of the absorption and emission spectra. The fluorescence decay times are generally higher than those of the monomers, and depend on the medium in which the planar aggregates are formed.

The photooxidation properties of porphyrin planar aggregates, as revealed by oxygen consumption and histidine photodegradation upon irradiation at 365 nm, were compared to those of the monomers. The extent of the photooxidation process is nearly 20−30 % higher in planar aggregates than in the monomers. In contrast, it is well known that cofacial aggregates are photochemically inert and only monomeric species of porphyrin are efficient photosensitizers. The biological relevance of these findings is discussed.
Structure of the O‐antigen of Vibrio cholerae O155 that shares a putative	D‐galactose 4,6‐cyclophosphate‐associated epitope with V. cholerae O139 Bengal
Authors: Senchenkova Sof'ya N. , Zatonsky Georgy V. , Shashkov Alexander S. , Knirel Yuriy A. , Jansson Per‐Erik , Weintraub Andrej , Albert M. John
Published: March 2003

The O‐specific polysaccharide of Vibrio cholerae O155 was studied by sugar and methylation analyses, dephosphorylation with 48 % hydrofluoric acid, 1H‐ and 13C‐NMR spectroscopy, including two‐dimensional COSY, TOCSY, NOESY, and heteronuclear single‐quantum coherence (HSQC) experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established :
HO
O
@dU
@dR@dR
P
@dR
@dU
4
6
α‐
D‐Galp‐(1←3)‐α‐
L‐Fucp‐(1←4)‐α‐
D‐GlcpNAc
1
↓
3
←3)‐β‐
D‐FucpNAc‐(1←4)‐α‐
L‐Fucp‐(1←   An unusual component,
D‐galactose 4,6‐cyclophosphate, has been reported previously as a component of the capsular polysaccharide and O‐antigen of V. cholerae O139 Bengal and appears to be responsible for the known serological cross‐reactivity between V. cholerae O139 and O155.
Transferrins
Authors: Pakdaman Rowchanak , Petitjean Michel , El Hage Chahine Jean‐Michel
Published: March 2003

Iron uptake by bovine lactoferrin from nitrilotriacetatoFe(III) [FeN(Ac)3] in the presence of bicarbonate has been investigated at pH 7.1−8.7. Deprotonated apolactoferrin interacting with bicarbonate or carbonate extracts iron from nitrilotriacetatoFe(III); the direct second‐order rate constant k1 = (4.90 ± 0.20)×104 M−1 s−1, a reverse second‐order rate constant k−1 = (1.80 ± 0.05)×105 M−1 s−1, and the iron‐exchange equilibrium constant K1 = 0.25 ± 0.05. The newly formed iron‐protein complex loses a single proton with proton dissociation constant K3a = (17 ± 0.5) nM, then undergoes a modification in its conformation followed by the loss of two or three protons; the first‐order rate constant k2 = (1.0 ± 0.10) s−1. This induces a new modification in the conformation; the first‐order rate constant k3 = (8.75 ± 0.40)×10−3 s−1. This second modification in conformation controls the rate of iron uptake by the N site of the protein and is followed by a single proton loss; K5a = 8.0 nM. Finally, the holoprotein or the monoferric lactoferrin in their final equilibrated states are produced by a third modification in the conformation occurring in about 9000 s. The mechanism of iron uptake by lactoferrin is very similar to that of serum transferrin with a cooperativity between the C and N sites upon iron uptake but with lower rates, higher affinities and at least one more proton loss involved. These differences may be the result of slight discrepancies in the intimate structures of binding sites for serum transferrin and lactoferrin. In order to analyse the cooperativity between these iron‐binding sites, the three‐dimensional position of the chain of amino acid residues separating the N and C lobes of human apo‐, holo‐ and dicopper‐lactoferrin have been compared by the recognition of the three‐dimensional shape dissimilarity program. The interlobe peptides of human hololactoferrin and apolactoferrin showed only 75.5 % tridimensional similarity, indicating that iron uptake affects the three‐dimensional structure of the interlobe chain.
Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate
Authors: Christeller John T. , Farley Peter C. , Ramsay Rochelle J. , Sullivan Patrick A. , Laing William A.
Published: March 2003

Phloem exudate from squash fruit contains heat‐inactivated material which inhibits pepsin activity. This inhibitory activity was purified by mild acid treatment, chromatography on trypsin‐agarose, Sephadex G‐75 and reverse‐phase HPLC, resulting in the elution of three peaks with pepsin‐inhibitory activity. N‐terminal sequencing indicated a common sequence of MGPGPAIGEVIG and the presence of minor species with seven‐ or two‐amino‐acid N‐terminal extensions beyond this point. Microheterogeneity in this end sequence was exhibited within and between two preparations. Internal sequencing of a major peak after a trypsin digestion gave the sequence FYNVVVLEK. The common N‐terminal sequence was used to design a degenerate primer for 3′ rapid amplification of cDNA ends and cDNA clones encoding two isoforms of the inhibitor were obtained. The open reading frames of both cDNAs encoded proteins (96 % identical) which contained the experimentally determined internal sequence. The amino acid content calculated from the predicted amino acid sequence was very similar to that measured by amino acid analysis of the purified inhibitor. The two predicted amino acid sequences (96 residues) had neither similarity to any other aspartic proteinase inhibitor nor similarity to any other protein. The inhibitors have a molecular mass of 10 552 Da, measured by matrix‐assisted laser‐desorption ionisation time‐of‐flight mass spectrometry and approximately 10 000 Da by SDS/PAGE, and behave as dimers of approximately 21 000 Da during chromatography on Superdex G‐75 gel‐filtration medium. The calculated molecular masses from the predicted amino acid sequences were 10 551 Da and 10 527 Da. The inhibitor was capable of inhibiting pepsin (Ki = 2 nM) and a secreted aspartic proteinase from the fungus Glomerella cingulata (Ki = 20 nM). The inhibitor, which is stable over acid and neutral pH, has been named squash aspartic proteinase inhibitor (SQAPI).