bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase
Authors: Bycroft Matthew , Weissman Kira J. , Staunton James , Leadlay Peter F.
Published: March 2003

Modular polyketide synthases (PKSs), such as the 6‐deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3–4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady‐state kinetic analysis of a typical bimodular PKS, 6‐deoxyerythronolide B synthase 1‐thioesterase (DEBS 1‐TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high‐yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size‐exclusion chromatography. The method provides 13‐fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl‐CoA hydrolase activity, which competes with polyketide chain extension.

The most reliable value for the kcat for synthesis of (3S,5R)‐dihydroxy‐(2R,4R)‐dimethyl‐n‐heptanoic acid‐δ‐lactone is 0.84 min−1, and the apparent Km for (2RS)‐methylmalonyl‐CoA is 17 µm. This kcat is approximately 10‐fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1‐TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.
Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A
Authors: Klink Tony A. , Woycechowsky Kenneth J. , Taylor Kimberly M. , Raines Ronald T.
Published: March 2003

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124‐residue enzyme that contains four interweaving disulfide bonds (Cys26–Cys84, Cys40–Cys95, Cys58–Cys110, and Cys65–Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild‐type RNase A and each disulfide variant unfold in a two‐state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino‐acid sequence (Cys26–Cys84 and Cys58–Cys110) enhance stability more than do the two embedded ones (Cys40–Cys95 and Cys65–Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 °C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2‐ to 40‐fold less than that of wild‐type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.
Structure of a glycerol teichoic acid‐like O‐specific polysaccharide of Proteus vulgaris O12
Authors: Perepelov Andrei V. , Torzewska Agnieszka , Shashkov Alexander S. , Senchenkova Sof’ya N. , Rozalski Antoni , Knirel Yuriy A.
Published: March 2003

A phosphorylated O‐specific polysaccharide (O‐antigen) was obtained by mild acid degradation of Proteus vulgaris O12 lipopolysaccharide and studied by sugar and methylation analyses, 1H‐, 13C‐ and 31P‐NMR spectroscopy, including two‐dimensional COSY, TOCSY, NOESY, H‐detected 1H, 13C and 1H, 31P heteronuclear multiple‐quantum coherence experiments. It was found that the polysaccharide consists of pentasaccharide repeating units connected via a glycerol phosphate group, and has the following structure:

where FucNAc is 2‐acetamido‐2,6‐dideoxygalactose and the degree of O‐acetylation at position 4 of GalNAc is ≈ 25%. Immunochemical studies with P. vulgaris O12 O‐antiserum suggested that the lipopolysaccharide studied shares common epitopes with the lipopolysaccharide core of P. vulgaris O8 and with the O‐antigens of P. penneri strains 8 and 63.
Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding
Authors: Kintrup Martin , Schubert Peter , Kunz Michael , Chabbert Marie , Alberti Patricia , Bombarda Elisa , Schneider Siegfried , Hillen Wolfgang
Published: March 2003

We analysed the conformational states of free, tet operator‐bound and anhydrotetracycline‐bound Tet repressor employing a Trp‐scanning approach. The two wild‐type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162–173, representing part of an unstructured loop and the N‐terminal six residues of α‐helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline‐binding constants were decreased up to 7.5‐fold when Trp was in positions 169, 170 and 173. Helical positions (168–173) differed from those in the loop (162–167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator‐complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline‐bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent‐accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline‐bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in α‐helix 9 become more buried upon the induction of TetR by anhydrotetracycline.
Functional characteristics of two BKCa channel variants differentially expressed in rat brain tissues
Authors: Ha Tal Soo , Jeong Soon Youn , Cho Suk‐Woo , Jeon Hyun‐kyu , Roh Gu Seob , Choi Wan Sung , Park Chul‐Seung
Published: March 2003

cDNAs encoding large‐conductance Ca2+‐activated K+ channel α‐subunit (rSlo) were obtained from rat brain. From the DNA sequence of multiple rslo clones, we identified a specific sequence variation of 81 nucleotides, which is either absent from or present at the N‐terminal region of a putative Ca2+‐sensing domain of the channel. Transcripts containing such variations were detected in different ratios from several brain regions, and their functional significance was further examined. When heterologously expressed in Xenopus oocytes, both rSlo variants, named rSlo0 and rSlo27, generated Ca2+‐activated and voltage‐activated K+ currents characteristic of neuronal large‐conductance Ca2+‐activated K+ (BKCa) channels. Single‐channel recordings of the two channels showed almost identical permeation characteristics and steady‐state gating behavior. Noticeable differences between rSlo0 and rSlo27 were revealed when the macroscopic currents were measured at various voltages and intracellular Ca2+ concentrations. rSlo27 activated was more rapidly than rSlo0 in the presence of the same voltage stimulus, and the differences in these activation kinetics were dependent on the concentration of intracellular Ca2+. Despite their similar apparent affinities for Ca2+, rSlo0 and rSlo27 showed significant differences in their co‐operative gating behavior. The Hill coefficient for intracellular Ca2+ was estimated to be about 3.7 for rSlo27 regardless of the membrane voltage, and that for rSlo0 was reduced from about 5 to 2 as the membrane voltage changed from 40 to 140 mV. As activation of BKCa channels is involved in rapid hyperpolarization of action potentials, the differential processing of rslo transcripts, and the generation of channels with different activation kinetics and Ca2+ cooperativity may be a mechanism for tuning the excitability of neurons in different brain regions.
Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis
Authors: Bushell Martin , Wood Wendy , Clemens Michael J. , Morley Simon J.
Published: March 2003

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase‐3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the α subunit of eIF2 and the eIF4E‐binding protein 4E‐BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2β and eIF2γ levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase‐3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E‐BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.
Specificity of the wound‐induced leucine aminopeptidase (LAP‐A) of tomato
Authors: Gu Yong‐Qiang , Walling Linda L.
Published: March 2003

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP‐A) that preferentially hydrolyzes amino acid‐p‐nitroanilide and ‐β‐naphthylamide substrates with N‐terminal Leu, Met and Arg residues. To determine the substrate specificity of LAP‐A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined. For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel. Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed. Substrates with variable N‐terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1′) position. The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N‐terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur‐containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides. Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted. To systematically evaluate the impact of the P1′ residue on cleavage of dipeptides, three series of dipeptides (Leu‐Xaa, Gly‐Xaa, and Arg‐Xaa) were evaluated. The P1′ residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue. P1′ Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP‐A, porcine LAP, and E. coli PepA markedly. Analysis six Arg‐Gly‐Xaa tripeptides showed that more diversity was tolerated in the P2′ position. P2′ Arg inhibited tripeptide cleavage by all three enzymes, while P2′ Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs.
Diverting a protein from its cellular location by intracellular antibodies
Authors: Lener Matteo , Horn Ivo R. , Cardinale Alessio , Messina Samantha , Nielsen U. B. , Rybak Susanne M. , Hoogenboom Hennie R. , Cattaneo Antonino , Biocca Silvia
Published: March 2003

We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single‐chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K‐Ras transformed cells all the anti‐Ras single‐chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti‐Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage‐derived anti‐Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.
Molecular characterization of two genes from Streptomyces tendae Tü901 required for the formation of the 4‐formyl‐4‐imidazolin‐2‐one‐containing nucleoside moiety of the peptidyl nucleoside antibiotic nikkomycin
Authors: Lauer Bettina , Russwurm Roland , Bormann Christiane
Published: March 2003

The genes nikQ and nikR were identified by sequencing DNA of the nikkomycin biosynthetic gene cluster from Streptomyces tendae Tü901/8c. The nikQ gene encodes a P450 cytochrome, and the predicted NikR gene product shows 48–56% sequence identity with uracil phosphoribosyltransferases from eukaryotic organisms. The nikQ and nikR genes were inactivated separately by insertion of a kanamycin‐resistance cassette. Inactivation of the nikQ gene abolished synthesis of nikkomycins containing 4‐formyl‐4‐imidazolin‐2‐one as the base (nikkomycins X and I), whereas production of nikkomycins containing uracil (nikkomycins Z and J) was not affected. Nikkomycin X and I production could be restored by feeding 4‐formyl‐4‐imidazolin‐2‐one to the nikQ mutants, indicating that NikQ is responsible for its formation from l‐histidine. Disruption of the nikR gene resulted in formation of decreased amounts of nikkomycins X and I, whereas nikkomycins Z and J were synthesized at wild‐type levels. A fluorouracil‐resistant nikR mutant lacking uracil phosphoribosyltransferase (UPRTase) activity did not synthesize nikkomycins X and I and accumulated 4‐formyl‐4‐imidazolin‐2‐one in its culture filtrate, whereas formation of nikkomycins Z and J was unimpaired. The mutant was complemented to nikkomycin X and I production by nikR expressed from the mel promoter of plasmid pIJ702. The nikR gene expressed in Escherichia coli led to the production of UPRTase activity. Our results indicate that NikR converts 4‐formyl‐4‐imidazolin‐2‐one to yield 5′‐phosphoribosyl‐4‐formyl‐4‐imidazolin‐2‐one, the precursor of nikkomycins containing this base.
The NMR solution structure of the ion channel peptaibol chrysospermin C bound to dodecylphosphocholine micelles
Authors: Anders Roswitha , Ohlenschläger Oliver , Soskic Vukic , Wenschuh Holger , Heise Bert , Brown Larry R.
Published: March 2003

Chrysospermin C is a 19‐residue peptaibol capable of forming transmembrane ion channels in phospholipid bilayers. The conformation of chrysospermin C bound to dodecylphosphocholine micelles has been solved using heteronuclear NMR spectroscopy. Selective15N‐labeling and13C‐labeling of specific α‐aminoisobutyric acid residues was used to obtain complete stereospecific assignments for all eight α‐aminoisobutyric acid residues. Structures were calculated using 339 distance constraints and 40 angle constraints obtained from NMR data. The NMR structures superimpose with mean global rmsd values to the mean structure of 0.27 Å (backbone heavy atoms) and 0.42 Å (all heavy atoms). Chrysospermin C bound to decylphosphocholine micelles displays two well‐defined helices at the N‐terminus (residues Phe1‐Aib9) and C‐terminus (Aib13‐Trp‐ol19). A slight bend preceding Pro14, i.e. encompassing residues 10–12, results in an angle of ≈ 38° between the mean axes of the two helical regions. The bend structure observed for chrysospermin C is compatible with the sequences of all 18 long peptaibols and may represent a common ‘active’ conformation. The structure of chrysospermin C shows clear hydrophobic and hydrophilic surfaces which would be appropriate for the formation of oligomeric ion channels.
Complexes of smooth muscle tropomyosin with F‐actin studied by differential scanning calorimetry
Authors: Levitsky Dmitrii I. , Rostkova Elena V. , Orlov Victor N. , Nikolaeva Olga P. , Moiseeva Ljudmila N. , Teplova Marianna V. , Gusev Nikolai B.
Published: March 2003

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal‐muscle F‐actin. In the absence of F‐actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 °C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F‐actin causes a 2–6 °C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F‐actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well‐separated peaks of tropomyosin were observed on the thermogram besides the peak of F‐actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45–47 °C corresponding to tropomyosin bound to F‐actin. By measuring the temperature‐dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F‐actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F‐actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F‐actin, which denatures with a maximum at 64 °C in the absence and at 78 °C in the presence of a twofold molar excess of phalloidin. After the F‐actin–tropomyosin complex had been heated to 90 °C and then cooled (i.e. after complete irreversible denaturation of F‐actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F‐actin and actin‐bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F‐actin to be probed very precisely.
Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491
Authors: Andrade Susana L. A. , Brondino Carlos D. , Feio Maria J. , Moura Isabel , Moura José J. G.
Published: March 2003

A novel molybdenum iron‐sulfur‐containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate‐reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97‐kDa homodimer, with 0.6 ± 0.1 Mo, 3.6 ± 0.1 Fe and 0.9 ± 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis–Menten kinetics, with the following parameters (for benzaldehyde): K  appm = 6.65 µm; V  app = 13.12 µm·min−1; k  appcat = 0.96 s−1. Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe–2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of −275 and −325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR‐distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.
Toxicity of non‐Aβ component of Alzheimer's disease amyloid, and N‐terminal fragments thereof, correlates to formation of β‐sheet structure and fibrils
Authors: Bodles Angela M. , Guthrie David J. S. , Harriott Patrick , Campbell Pamela , Irvine G. Brent
Published: March 2003

The non‐Aβ component of Alzheimer's disease amyloid (NAC) and its precursor α‐synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms β‐sheet structures and fibrils [El‐Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem.258, 157–163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 °C the full‐length peptide and fragments NAC(3–18) and NAC(1–18) scrambled sequence [NAC(1–18 s)] were toxic, whereas fragments NAC(1–13) and NAC(6–14) were not. CD indicates that NAC(3–18) and NAC(1–18 s) exhibit β‐sheet secondary structure in aqueous solution, whereas NAC(1–13) and NAC(6–14) do not. NAC(3–18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1–18 s) it does not come out of solution to a significant degree. Fragments NAC(1–13) and NAC(6–14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form β‐sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Aβ peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying β‐sheet. These results suggest that an intermediate, which exhibits β‐sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.
A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily
Authors: Castillo Josefa , Rodrigo M. Isabel , Márquez José A. , Zúñiga Ángel , Franco Luis
Published: March 2003

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4‐kDa polypeptide (p54). p16 corresponds to the C‐terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose‐binding soybean protein, and, to a lesser extent (31–34%), it shares homology with some storage proteins. p16 is also 30% homologous with Nhp2p, a yeast nuclear protein. The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation. Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation). p16 can be recovered from nuclei cross‐linked to histone H3, when the disulfide bridges that occur in vivo are preserved. On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation. We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles. The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.
Structural analysis of the core region of lipopolysaccharides from Proteus mirabilis serotypes O6, O48 and O57
Authors: Vinogradov Evgeny , Perry Malcolm B.
Published: March 2003

The structure of lipid A core region of the lipopolysaccharides (LPS) from Proteus mirabilis serotypes O6, O57 and O48 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of LPS:

Incomplete substitutions are indicated by bold italic type. All sugars are present in pyranose form, α‐Hep is the residue of lglycero‐α‐dmanno‐Hep, α‐dd‐Hep is the residue of dglycero‐α‐dmanno‐Hep, l‐Ara4N is 4‐amino‐4‐deoxy‐l‐arabinose, Qui4NAlaAla is the residue of 4‐N‐(l‐alanyl‐l‐alanyl)‐4‐amino‐4,6‐dideoxyglucose. All sugars except l‐Ara4N have d‐configuration. β‐GalA* is partially present in the form of amide with 1,4‐diaminobutane (putrescine)‐HN(CH2)4NH2 or spermidine‐HN(CH2)3NH(CH2)4NH2.
Characterization of the early steps of OE17 precursor transport by the thylakoid ΔpH/Tat machinery
Authors: Musser Siegfried M. , Theg Steven M.
Published: March 2003

In order to probe the structure and protein translocation function of the thylakoid Tat machinery, a 25‐residue C‐terminal extension containing a 13‐residue in vivo biotinylation tag and a 6× His tag was added to a mutant precursor of the 17‐kDa subunit of the oxygen‐evolving complex to form pOE17(C)‐BioHis. When avidin was attached to biotinylated precursor in situ, the precursor–avidin complex was neither imported nor did it form a membrane‐spanning translocation intermediate. It did, however, competitively inhibit the translocation of unbiotinylated precursor with an apparent KI unaffected by avidin. It is shown that the precursor protein achieves a stable folded structure upon dilution from urea, suggesting that the avidin‐induced inhibition of transport results from a folding‐induced proximity of N‐terminal and C‐terminal domains. It is further demonstrated that the majority of precursor rapidly binds to the thylakoid membrane, remaining import competent and yet undissociable by high salt or high pH treatment at ice temperature. The membrane binding event is unaffected by avidin. Import kinetics reveal that nonproton motive force‐driven transport steps make up a major fraction of the transport time. These observations suggest that the N‐terminal presequence on the avidin‐bound precursor is available for membrane binding and initial recognition by the transport machinery, but the attached avidin signals the machinery that the precursor is an incorrectly configured substrate and thus import is aborted. Consequently, the ΔpH/Tat machinery's proofreading mechanism must operate after precursor recognition but before the committed step in transport.
Shedding of interleukin‐6 receptor and tumor necrosis factor α
Authors: Althoff Katja , Reddy Pranitha , Voltz Nicole , Rose‐John Stefan , Müllberg Jürgen
Published: March 2003

A functionally and structurally diverse group of transmembrane proteins including transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteolytic activity responsible for pro‐tumor necrosis factor α (proTNFα) processing has been identified and named TACE (TNFα converting enzyme). In experiments with TACE deficient (TACE‐/‐) fibroblasts we found that 4β‐phorbol 12‐myristate 13‐acetate (PMA)‐induced shedding of the interleukin‐6 receptor (IL‐6R) is strongly reduced. A basal hydroxamate sensitive release of IL‐6R, however, could still be detected. This result demonstrates that TACE plays a role in IL‐6R processing and that additional metalloproteases might be involved. PMA‐induced shedding of IL‐6R in TACE deficient mouse fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL‐6R and proTNFα we generated chimeric IL‐6R and proTNFα proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNFα and IL‐6R, respectively. Interestingly, proTNFα chimeric proteins showed only minimal shedding. In contrast, IL‐6R chimeras containing the proTNFα CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNFα CS is similar to that of proTNFα itself. We conclude that the amino‐acid sequence at the proteolytic CS contributes to the cleavage characteristics of a protein. However, this information alone is not sufficient to transfer cleavability as seen with proTNFα chimeras containing the IL‐6R CS and which were resistant to shedding.
Flexibility and stability of the structure of cytochromes P450 3A4 and BM‐3
Authors: Anzenbacherová Eva , Bec Nicole , Anzenbacher Pavel , Hudeček Jiří , Souček Pavel , Jung Christiane , Munro Andrew W. , Lange Reinhard
Published: March 2003

The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM‐3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun.252, 166–172].
Site‐directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B
Authors: Zhang Sheng , Irwin Diana C. , Wilson David B.
Published: March 2003

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2,4‐dinitrophenyl‐β‐d‐cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites −2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild‐type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.
Structure of the sialic acid‐containing O‐specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide
Authors: Gamian Andrzej , Jones Christopher , Lipiński Tomasz , Korzeniowska‐Kowal Agnieszka , Ravenscroft Neil
Published: March 2003

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 °C or 0.1 m NaOH‐AcOH, pH 4.5, 5 h, 100 °C), the O‐specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O‐specific polysaccharide was shown to be the following trisaccharide:

→4)‐α‐Neup5Ac(2→3)‐l‐α‐FucpNAc(1→3)‐d‐β‐GlcpNAc(1→

The polysaccharide O‐chain was substituted with a single molar equivalent of O‐acetyl group, distributed between the Neu5Ac O‐9 and O‐7 positions, in an approximate ratio of 7 : 3.
The oxidation and reduction reactions of bovine serum amine oxidase
Authors: Bellelli Andrea , Morpurgo Laura , Mondovì Bruno , Agostinelli Enzo
Published: March 2003

The presteady‐state and steady‐state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped‐flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady‐state parameters.

The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry37, 12513–12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured.

The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate.

These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans‐iminative proton abstraction occurring in the enzyme–substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine‐radical intermediate, while the latter is less easily interpreted.
Physicochemical characteristics of triacyl lipid A partial structure OM‐174 in relation to biological activity
Authors: Brandenburg Klaus , Lindner Buko , Schromm Andra , Koch Michel H. J. , Bauer Jacques , Merkli Alain , Zbaeren Claude , Davies J. Gwynfor , Seydel Ulrich
Published: March 2003

The triacylated lipid A partial structure OM‐174 was characterized in detail using a variety of physical and biological techniques. OM‐174 aggregates adopt the micellar HI structure. The temperature (Tc) of the gel to liquid‐crystalline phase transition of the hydrocarbon chains is 0 °C, from which high fluidity of the acyl chains at 37 °C can be deduced. The molecular area of a single OM‐174 molecule at a surface pressure of 30 mN·m−1 is 0.78 ± 0.04 nm2. Conformational analyses, using IR spectroscopy, of the behavior of the various functional groups of OM‐174 as compared with hexa‐acyl lipid A suggest altered hydration of the phosphate charges and unusually strong hydration of the ester groups, which is probably related to the high accessibility of these groups to water in the micellar aggregate structure. OM‐174 was shown to intercalate into a phospholipid membrane corresponding to the macrophage membrane within seconds in the presence, and within minutes to hours in the absence, of LPS‐binding protein. In the Limulus amebocyte lysate assay, the triacyl lipid A is more than 105‐fold less active than hexa‐acyl lipid A, but only 10‐fold less active in inducing IL‐6 in human mononuclear cells, and equally active in inducing NO production in murine macrophages. These findings are used to explain the mechanism of the lipid A‐induced cell activation.
Archaeal protein translocation
Authors: Eichler Jerry
Published: March 2003

Proper cell function relies on correct protein localization. As a first step in the delivery of extracytoplasmic proteins to their ultimate destinations, the hydrophobic barrier presented by lipid‐based membranes must be overcome. In contrast to the well‐defined bacterial and eukaryotic protein translocation systems, little is known about how proteins cross the membranes of archaea, the third and most recently described domain of life. In bacteria and eukaryotes, protein translocation occurs at proteinaceous sites comprised of evolutionarily conserved core components acting in concert with other, domain‐specific elements. Examination of available archaeal genomes as well as cloning of individual genes from other archaeal strains reveals the presence of homologues to selected elements of the bacterial or eukaryotic translocation machines. Archaeal genomic searches, however, also reveal an apparent absence of other, important components of these two systems. Archaeal translocation may therefore represent a hybrid of the bacterial and eukaryotic models yet may also rely on components or themes particular to this domain of life. Indeed, considering the unique chemical composition of the archaeal membrane as well as the extreme conditions in which archaea thrive, the involvement of archaeal‐specific translocation elements could be expected. Thus, understanding archaeal protein translocation could reveal the universal nature of certain features of protein translocation which, in some cases, may not be readily obvious from current comparisons of bacterial and eukaryotic systems. Alternatively, elucidation of archaeal translocation could uncover facets of the translocation process either not yet identified in bacteria or eukaryotes, or which are unique to archaea. In the following, the current status of our understanding of protein translocation in archaea is reviewed.
Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin‐like protein in the presence of dopamine
Authors: Lee Kwang Moon , Lee Kum Young , Choi Hye Won , Cho Mi Young , Kwon Tae Hyuk , Kawabata Shun‐ichiro , Lee Bok Luel
Published: March 2003

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G‐100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and β‐1,3‐glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G‐100 column. When G‐100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and β‐1,3‐glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G‐100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization‐engaging proteins, we first purified the 160‐kDa melanization‐engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino‐acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor‐6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160‐kDa melanization‐engaging protein was added to a G‐100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160‐kDa vitellogenin‐like protein is involved in arthropod melanin synthesis.
Export and folding of signal‐sequenceless Bacillus licheniformisβ‐lactamase in Escherichia coli
Authors: Frate María C. , Lietz Eric J. , Santos Javier , Rossi Juan P. F. C. , Fink Anthony L. , Ermácora Mario R.
Published: March 2003

Two genetically engineered variants of the Bacillus licheniformisβ‐lactamase gene were expressed in Escherichia coli. One variant coded for the exo‐small mature enzyme without the signal peptide. The other coded for the exo‐large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme‐EDTA treatment, the signal‐less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N‐terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal‐less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large ΔG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non‐native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coliβ‐lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.
The PKC targeting protein RACK1 interacts with the Epstein–Barr virus activator protein BZLF1
Authors: Baumann Matthias , Gires Olivier , Kolch Walter , Mischak Harald , Zeidler Reinhard , Pich Dagmar , Hammerschmidt Wolfgang
Published: March 2003

Phorbol esters reactivate Epstein–Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate‐early gene BZLF1. BZLF1 is a member of the extended AP‐1 family of transcription factors that binds to specific BZLF1‐binding motifs within early EBV promoters and to consensus AP‐1 sites. Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post‐translational modifications. Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA [Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H. J. & Hammerschmidt, W., (1998) J. Virol.72, 8105–8114]. The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay. RACK stands for receptor of activated C‐kinase and is involved in targeting activated PKCs and other signaling proteins. In vivo, RACK1 binds directly to the transactivation domain of BZLF1. Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays. We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs.
Structure and expression of mGDF, a new member of the transforming growth factor‐β superfamily in the bivalve mollusc Crassostrea gigas
Authors: Lelong Christophe , Mathieu Michel , Favrel Pascal
Published: March 2003

To gain insight into the evolution of the structure and functions of transforming growth factor (TGF)‐β superfamily members, a cDNA encoding a new member from the bivalve mollusc Crassostrea gigas named mGDF (molluscan growth and differentiation factor) was identified by PCR using degenerate primers. The mGDF precursor exhibits characteristic features of the TGF‐β superfamily and shows highest homology with human BMP2 and Drosophila DPP. Conversely, the mgdf gene displays a distinct pattern of expression during development. Indeed mgdf transcripts were not detected early in development but increased markedly before metamorphosis. These findings raise the possibility that mGDF could play a central role in the biological processes that allow larvae to become competent to metamorphose.