bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA hydratase, an enzyme involved in anaerobic metabolism of benzoyl‐CoA in the denitrifying bacterium Thauera aromatica
Authors: Laempe Diana , Eisenreich Wolfgang , Bacher Adelbert , Fuchs Georg 
Published: March 2003

Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl‐CoA as the common intermediate. The central pathway of benzoyl‐CoA metabolism is initiated by an ATP‐driven reduction of the aromatic ring producing cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA. The 1,5‐dienoyl‐CoA intermediate is thought to be transformed to 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA by a specific dienoyl‐CoA hydratase catalyzing the formal addition of water to one of the double bonds.This dienoyl‐CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate. Substrate and product were confirmed and a convenient spectrophotometric assay was developed. The equilibrium concentrations of substrate and product were almost equal. Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate. The enzyme of 28 kDa was purified from T. aromatica and was found to be highly specific for the cyclic 1,5‐dienoyl‐CoA. A second 29‐kDa enoyl‐CoA hydratase acted on crotonyl‐CoA; this highly active enoyl‐CoA hydratase also acted slowly on cyclohex‐1‐ene‐1‐carboxyl‐CoA. The regulation of expression of dienoyl‐CoA hydratase activity, the kinetic constants, the substrate specificity, and the specific activity of the enzyme in cell extract provide evidence that dienoyl‐CoA hydratase is the second enzyme of the central benzoyl‐CoA pathway of anaerobic aromatic metabolism in T. aromatica.Extracts of Rhodopseudomonas palustris contained high activity of cyclohex‐1‐ene‐1‐carboxyl‐CoA hydratase, but no 1,5‐dienoyl‐CoA hydratase activity. It appears that a variant of the benzoyl‐CoA pathway is operating in R. palustris in which hydration of the 1,5‐dienoyl‐CoA does not take place. Rather, cyclohex‐1‐ene‐1‐carboxyl‐CoA is hydrated to 2‐hydroxycyclohexane‐1‐carboxyl‐CoA.
Structural elucidation of the lipopolysaccharide core regions of the wild‐type strain PAO1 and O‐chain‐deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5
Authors: Sadovskaya Irina , Brisson Jean‐Robert , Lam Joseph S. , Richards James C. , Altman Eleonora 
Published: March 2003

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild‐type strain PAO1 and derived rough‐type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum‐ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of
D‐glucose (four units),
L‐rhamnose (one unit), 2‐amino‐2‐deoxy‐
D‐galactose (one unit),
Lglycero‐
D‐manno‐heptose (Hep; two units), 3‐deoxy‐
D‐manno‐octulosonic acid (Kdo; two units),
Lalanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one‐dimensional and two‐dimensional (2D) NMR techniques in combination with MS‐based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two‐dimensional 31P (ω1)‐half‐filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild‐type strain. The presence of a 7‐O‐carbamoyl substituent was observed on the second heptose. The structure of the core region of the O‐chain‐deficient LPS from P. aeruginosa serotype O5 is as follows :
R2
CONH2P↓↓↓
3
7
6
R1←4)‐α‐
D‐GalpN‐(1←3)‐
L‐α‐
D‐Hepp‐(1←3)‐
L‐α‐
D‐Hepp‐(1←5)‐α‐
D‐Kdop‐(2←6)‐lipid A
2
2
4
4
↑↗↖↑L‐Ala
P
P
2
α‐
D‐Kdop
where R1 is β‐
D‐Glcp‐(1←2)‐α‐
L‐Rhap‐(1←6)‐α‐
D‐Glcp‐(1← and R2 is α‐
D‐Glcp‐(1←6)‐β‐
D‐Glcp‐(1←.A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.
Elucidation of the structure of an alanine‐lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4
Authors: Sánchez Carballo Patricia M. , Rietschel Ernst Th. , Kosma Paul , Zähringer Ulrich 
Published: March 2003

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough‐type LPS. Additional faint high molecular‐mass bands revealed that the O‐chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel‐permeation chromatography and high‐performance anion‐exchange liquid chromatography. The oligosaccharide contained two residues of lglycerodmanno‐heptose (Hep) and one residue each of 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid (Kdo) and GalNAc. Upon matrix‐assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M‐H]− at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one‐ and two‐dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide:imageTwo unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide‐linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.
High prevalence of 2‐mono‐ and 2,6‐di‐substituted Manol‐terminating sequences among O‐glycans released from brain glycopeptides by reductive alkaline hydrolysis
Authors: Chai Wengang , Yuen Chun‐Ting , Kogelberg Heide , Carruthers Robert A. , Margolis Richard U. , Feizi Ten , Lawson Alexander M. 
Published: March 2003

Di‐ to heptasaccharides isolated from total nondialyzable brain glycopeptides after release by alkaline borohydride treatment have been subjected to mass spectrometric and nuclear magnetic resonance spectroscopic analyses supplemented by TLC‐MS analyses of derived neoglycolipids. A family of Manol‐terminating oligosaccharides has been revealed which includes novel sequences with a 2,6‐disubstituted Manol:In contrast to the Manol‐terminating HNK‐1 antigen‐positive chains described previously that occur as a minor population [Yuen, C.‐T., Chai, W., Loveless, R.W., Lawson, A.M., Margolis, R.U. & Feizi, T. (1997) J. Biol. Chem.272, 8924–8931], the above oligosaccharides are abundant. The ratio of these compounds to the classical N‐acetylgalactosaminitol‐terminating oligosaccharides is about 1 : 3. Thus, there appears to be in higher eukaryotes a major alternative pathway related to the yeast‐type protein O‐mannosylation, the enzymatic basis and functional importance of which now require investigation.
Functional complementation analysis of yeastbc1mutants
Authors: van Wilpe Sandra , Boumans Hans , Lobo‐Hajdu Gisele , Grivell Leslie A. , Berden Jan A. 
Published: March 2003

Previous complementation studies with yeastbc1mutants, defective in subunit VII or VIII, using heterologous and hybrid subunits, suggested that the requirement for import into mitochondria might significantly restrict the scope of this test for compatible proteins. Prediction algorithms indicate that the N‐terminal domain of subunit VII contains all known characteristics of a mitochondrial targeting signal, whereas in subunit VIII such a signal is absent from the N‐terminal domain, but possibly present in an internal region of the protein. Despite the fact that the characteristics of a mitochondrial import signal are found in the N‐terminus of all known subunit‐VII orthologues,in vitroimport experiments show that the protein of human origin is not imported into yeast mitochondria.In vitroimport can be restored, however, by replacement of the N‐terminal part of the human protein by the N‐terminus of theSaccharomyces cerevisiaeorthologue, indicating a requirement for species‐specific elements. Similar experiments were performed with subunit VIII and orthologues thereof, including a hybrid protein in which the N‐terminus of the bovine heart orthologue was replaced by that ofS. cerevisiae. The ability of yeast mitochondria to import this hybrid protein, in contrast with the bovine subunit‐VIII orthologue itself, indicates that for subunit VIII also the N‐terminus, in contradiction of theoretical predictions, contributes to the targeting signal, most likely via species‐specific elements. Our findings expose the limitations of the currently available criteria for prediction of the presence and location of a mitochondrial targeting sequence and highlight the necessity of performing separate import studies for interpreting complementation studies as long as the species‐specific characteristics of the import signals have not been identified.
Expression, purification and copper‐binding studies of the first metal‐binding domain of Menkes protein
Authors: Jensen Pia Y. , Bonander Nicklas , Horn Nina , Tümer Zeynep , Farver Ole 
Published: March 2003

The cDNA, coding for the first metal‐binding domain (MBD1) of Menkes protein, was cloned into the T7‐system based vector, pCA. The T7 lysozyme‐encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg·L−1. Menkes protein, a P‐type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N‐terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)‐MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper‐bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 µm. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo–MBD1. Given the same total copper concentration CuCl43– was superior to Cu(I)–thiourea (structural analogue of metallothionein) and Cu(I)–glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo–MBD1. Cu(II) was not able to reconstitute apo–MBD1, presumably due to Cu(II)‐induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.
Reactive oxygen species released from granulocytes stimulate 5‐lipoxygenase activity in a B‐lymphocytic cell line
Authors: Werz Oliver , Szellas Dagmar , Steinhilber Dieter 
Published: March 2003

B‐lymphocytes express 5‐lipoxygenase (5‐LO) protein but cellular leukotriene production is suppressed by selenium‐dependent peroxidases. Thus it was of interest to check whether reactive oxygen species (ROS) which are released under inflammatory conditions can stimulate B‐lymphocyte 5‐LO and counteract peroxidase‐mediated suppression of cellular 5‐LO activity. It was found that 5‐LO in the Epstein–Barr virus‐transformed B‐lymphocytic cell line BL41‐E95‐A is activated by addition of hydrogen peroxide or xanthine/xanthine oxidase and after increasing the oxidative state of the cell by azodicarboxylic acid bis(dimethylamide). Generation of endogenous ROS from mitochondria by antimycin A also lead to a threefold upregulation of 5‐LO activity in B‐cells.There was almost no detectable endogenous superoxide formation in BL41‐E95‐A cells after stimulation with 4β‐phorbol 12‐myristate 13‐acetate. Co‐incubation experiments with BL41‐E95‐A cells and granulocytes demonstrated that granulocyte‐derived ROS can activate B‐lymphocyte 5‐LO. Addition of superoxide dismutase and/or catalase to the B‐lymphocyte/granulocyte co‐incubations and to B‐lymphocyte homogenates revealed that the 5‐LO activation is due to the superoxide‐derived release of hydroperoxides or hydrogen peroxide from granulocytes.The data suggest that ROS formation plays an important role in the regulation of cellular 5‐LO activity in B‐lymphocytes. As leukotrienes affect B‐cell functions like cell proliferation, activation and maturation, this finding provides a new link between the formation of ROS and the regulation of immune responses.
Characterization of five new low‐molecular‐mass trypsin inhibitors from white mustard (Sinapis alba L.) seed
Authors: Ruoppolo Margherita , Amoresano Angela , Pucci Piero , Pascarella Stefano , Polticelli Fabio , Trovato Maurizio , Menegatti Enea , Ascenzi Paolo 
Published: March 2003

Five new low‐molecular‐mass trypsin inhibitors belonging to the RTI/MTI‐2 family were identified from white mustard (Sinapis alba L.; MTI‐2) seed. Purified MTI‐2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C‐terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI‐2 inhibitors originated from different genes. The presence of 5‐oxo‐proline (pyroglutamic acid; 5‐oxoPro1) and Kyn44 reflected post‐translational processing of the serine proteinase inhibitor. MTI‐2 showed ≈ 70% amino‐acid identity with low‐molecular‐mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI‐III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI‐2 was homologous to brazzein, the sweet‐tasting protein from Pentadiplandra brazzeana Baillon fruit (≈ 30% amino‐acid identity). Although snake‐venom toxins showed a low amino‐acid identity (< 20%) with MTI‐2, RTI‐III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI‐2 (Cys5–Cys27, Cys18–Cys31, Cys42–Cys52, and Cys54–Cys57) corresponded to that of RTI‐III and of snake‐venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three‐dimensional arrangement rather than to the amino‐acid sequence. Values of Ka for MTI‐2 binding to bovine β‐trypsin (trypsin) and bovine α‐chymotrypsin were 6.3 × 109m−1 and 2.0 × 106 m−1, respectively, at pH 8.0 and 21.0 °C. Moreover, values of kon for MTI‐2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 × 105 m−1·s−1 and 8.9 × 10−5 m−1·s−1, respectively, at pH 8.0 and 21.0 °C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI‐2 inhibitors were indistinguishable.
Functions of transforming growth factor‐β isoforms in the nervous system
Authors: Unsicker Klaus , Strelau Jens 
Published: March 2003

This review briefly describes the cellular distribution and documented roles of the transforming growth factor (TGF)‐β isoforms TGF‐β2 and ‐β3 in the central and peripheral nervous system. TGF‐β2 and ‐β3 are coexpressed in developing radial glial and mature astroglial and Schwann cells, as well as in subpopulations of differentiated neurons, most prominently in cortical, hippocampal, and brainstem/spinal cord motor neurons. In vitro studies have suggested a number of potential, physiologically relevant functions for TGF‐βs including regulation of astroglial cell proliferation, expression of adhesion molecules, survival promoting roles for neurons in combination with established neurotrophic factors, and differentiative actions on neurons.
Methyl‐CpG‐binding proteins
Authors: Ballestar Esteban , Wolffe Alan P. 
Published: March 2003

CpG methylation, the most common epigenetic modification of vertebrate genomes, is primarily associated with transcriptional repression. MeCP2, MBD1, MBD2, MBD3 and MBD4 constitute a family of vertebrate proteins that share the methyl‐CpG‐binding domain (MBD). The MBD, consisting of about 70 residues, possesses a unique α/β‐sandwich structure with characteristic loops, and is able to bind single methylated CpG pairs as a monomer. All MBDs except MBD4, an endonuclease that forms a complex with the DNA mismatch‐repair protein MLH1, form complexes with histone deacetylase. It has been established that MeCP2, MBD1 and MBD2 are involved in histone deacetylase‐dependent repression and it is likely that this is also the case for MBD3. The current model proposes that MBD proteins are involved in recruiting histone deacetylases to methyl CpG‐enriched regions in the genome to repress transcription. The lack of selectivity for MBD association with particular DNA sequences indicates that other mechanisms account for their recruitment to particular regions in the genome.
Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins
Authors: Miller Richard W. , Eady Robert R. , Fairhurst Shirley A. , Gormal Carol A. , Smith Barry E. 
Published: March 2003

Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2′(3′)‐O‐[N‐methylanthraniloyl] ADP and AlF4− or BeF3− ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature387, 370–376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP‐binding sites of the iron protein, Kp2. The beryllium (BeFx) and aluminium (AlF4−) containing complexes are proposed to correspond to the ATP‐bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry34, 8960–8972]. 31P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the 31P NMR signals and, in the case of the AlF4− complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 ± 0.1 MgADP per mol Kp1. With the AlF4− complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF4− ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2′(3′)‐O‐[N‐methylanthraniloyl] ADP was blue‐shifted by 6–8 nm in both metal fluoride complexes and polarization was 6–9 times that of the free analogue. The fluorescence yield of bound 2′(3′)‐O‐[N‐methylanthraniloyl] ADP was enhanced by 40% in the AlF4− complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide‐binding pocket was marked in the AlF4− complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable 31P NMR; as detected in mature complexes with no detectable 31P NMR; in the AlF4− complex in which an altered tyrosine interaction permits resonance energy transfer with 2′(3′)‐O‐[N‐methylanthraniloyl] ADP.
Mutagenesis of key residues identifies the connection subdomain of HIV‐1 reverse transcriptase as the site of inhibition by heme
Authors: Argyris Elias G. , Vanderkooi Jane M. , Paterson Yvonne 
Published: March 2003

We have recently demonstrated that metalloporphyrins are potent inhibitors of both human immunodeficiency virus type 1 (HIV‐1) and human immunodeficiency virus type 2 (HIV‐2) reverse transcriptases (RTs) [Argyris, E.G., Vanderkooi, J.M., Venkateswaran, P.S., Kay, B.K., and Paterson, Y. (1999) J. Biol. Chem.274, 1549–1556]. In addition, by screening a phage peptide library we discovered that a peptide with sequence similarity to residues 398–407 from the connection subdomain of HIV RTs binds heme. These findings suggested that this highly conserved region may be the binding site for metalloporphyrins and a novel site for inhibition of enzymatic activity. Our most recent data presented here confirm this suggestion. Screening of HIV‐1 RT 398–407 peptide analogs by fluorescence assays demonstrates that Trp residues at positions 401 and 402 are important for heme binding. Furthermore, site‐directed mutagenesis of these residues verified these findings and indicated that heme inhibits HIV‐1 RT by binding on the connection subdomain of the p66 subunit of the enzyme but not on the p51 subunit. This was also confirmed by analyzing the binding affinities of heme for mutant HIV‐1 RT heterodimers, using intrinsic fluorescence assays. The clear identification of the connection domain as a novel inhibition site is crucial in understanding the mechanism of heme binding and enzymatic inhibition and will facilitate the generation of novel porphyrin‐based inhibitors of RT.
Characterization of oligosaccharides from the chondroitin sulfates
Authors: Huckerby Thomas N. , Lauder Robert M. , Brown Gavin M. , Nieduszynski Ian A. , Anderson Karen , Boocock Joanne , Sandall Patricia L. , Weeks Stephen D. 
Published: March 2003

Chondroitin sulfates were fragmented using the enzymes chondroitin sulfate ABC endolyase and chondroitin ACII lyase; both disaccharide and tetrasaccharide fragments were isolated after reduction to the corresponding 2‐deoxy‐2‐N‐acetylamino‐d‐galactitol (GalNAc‐ol) form. These have the structures: ΔUA(β1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc6S‐ol, ΔUA2S(β1–3)GalNAc6S‐ol, ΔUA(β1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc4S(β1–4)GlcA(β1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S‐ol, ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol, ΔUA2S(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S‐ol and ΔUA2S(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol, where ΔUA represents a 4,5‐unsaturated hexuronic acid (4‐deoxy‐α‐lthreo‐hex‐4‐enepyranosyluronic acid) and 6S/4S/2S represent O‐ester sulfate groups at C6/C4/C2 sites. Complete 1H‐NMR and 13C‐NMR data are derived for these species, which may help to alleviate some of the significant difficulties resulting from signal complexity that are currently hindering the characterization and assignment of major and minor structural components within chondroitin sulfate and dermatan sulfate polymers.