Biomedical subjects

A Böck

Publications and source records attributed to A Böck.

At least 37 records · Page 2Linked to original sources

PubMed1999

High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.

Mammalian thioredoxin reductase (TrxR) catalyzes reduction of thioredoxin and many other substrates, and is a central enzyme for cell proliferation and thiol redox control. The enzyme is a selenoprotein and can therefore, like all other mammalian selenoproteins, not be directly expressed in Escherichia coli, since selenocysteine-containing proteins are synthesized by a highly species-specific translation machinery. This machinery involves a secondary structure, SECIS element, in the selenoprotein-encoding mRNA, directing selenocysteine insertion at the position of an opal (UGA) codon, normally conferring termination of translation. It is species-specific structural features and positions in the selenoprotein mRNA of the SECIS elements that hitherto have hampered heterologous production of recombinant selenoproteins. We have discovered, however, that rat TrxR can be expressed in E. coli by fusing its open reading frame with the SECIS element of the bacterial selenoprotein formate dehydrogenase H. A variant of the SECIS element designed to encode the conserved carboxyterminal end of the enzyme (-Sec-Gly-COOH) and positioning parts of the SECIS element in the 3'-untranslated region was also functional. This finding revealed that the SECIS element in bacteria does not need to be translated for full function and it enabled expression of enzymatically active mammalian TrxR. The recombinant selenocysteine-containing TrxR was produced at dramatically higher levels than formate dehydrogenase O, the only endogenous selenoprotein expressed in E. coli under the conditions utilized, demonstrating a surprisingly high reserve capacity of the bacterial selenoprotein synthesis machinery under aerobic conditions. Co-expression with the selA, selB and selC genes (encoding selenocysteine synthase, SELB and tRNA(Sec), respectively) further increased the efficiency of the selenoprotein production and thereby also increased the specific activity of the recombinant TrxR to about 25 % of the native enzyme, with as much as 20 mg produced per liter of culture. These results show that with the strategy utilized here, the capacity of selenoprotein synthesis in E. coli is more than sufficient for making possible the use of the bacteria for production of recombinant selenoproteins.

3' Untranslated Regions↗

PubMed1999

Properties of a cyclodextrin-specific, unusual porin from Klebsiella oxytoca.

The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized. CymA is essential for growth on cyclodextrins, but it can also complement the deficiency of a lamB (maltoporin) mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates. CymA was overproduced in E. coli and purified to apparent homogeneity. CymA is a component of the outer membrane, is processed from a signal peptide-containing precursor, and possesses a high content of antiparallel beta-sheet. Incorporation of CymA into lipid bilayers and conductance measurements revealed that it forms ion-permeable channels, which exhibit a substantial current noise. CymA-induced membrane conductance decreased considerably upon addition of alpha-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K(S), of 28 microM for its binding to CymA. CymA assembled in vitro to two-dimensionally crystalline tubular membranes, which, on electron microscopy, are characterized by a p1-related two-sided plane group. The crystallographic unit cell contains four monomeric CymA molecules showing a central pore. The lattice parameters are a = 16.1 nm, b = 3.8 nm, gamma = 93 degrees. CymA does not form trimeric complexes in lipid membranes and shows no tendency to trimerize in solution. CymA thus is an atypical porin with novel properties specialized to transfer cyclodextrins across the outer membrane.

Amino Acid Sequence↗

PubMed1999

Crystal structure of the hydrogenase maturating endopeptidase HYBD from Escherichia coli.

The maturation of [NiFe] hydrogenases includes formation of the nickel metallocenter, proteolytic processing of the metal center carrying large subunit, and its assembling with other hydrogenase subunits. The hydrogenase maturating enzyme HYBD from Escherichia coli, a protease of molecular mass 17.5 kDa, specifically cleaves off a 15 amino acid peptide from the C terminus of the precursor of the large subunit of hydrogenase 2 in a nickel-dependent manner. Here we report the crystal structure of HYBD at 2.2 A resolution. It consists of a twisted five-stranded beta-sheet surrounded by four and three helices, respectively, on each side. A cadmium ion from the crystallization buffer binds to the proposed nickel-binding site and is penta-coordinated by Glu16, Asp62, His93, and a water molecule in a pseudo-tetragonal arrangement. HYBD is topologically related to members of the metzincins superfamily of zinc endoproteinases, sharing the central beta-sheet and three helices. In contrast to the metzincins, the metal-binding site of HYBD is localized at the C-terminal end of the beta-sheet. Three helical insertions unique to HYBD pack against one side of the sheet, build up the active site cleft, and provide His93 as ligand to the metal. From this structure, we derive molecular clues into how the protease HYBD is involved in the hydrogenase maturation process.

Amino Acid Sequence↗

PubMed1999

Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome.

The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.

Bacterial Proteins↗

PubMed1999

A family of S-methylmethionine-dependent thiol/selenol methyltransferases. Role in selenium tolerance and evolutionary relation.

Several plant species can tolerate high concentrations of selenium in the environment, and they accumulate organoselenium compounds. One of these compounds is Se-methylselenocysteine, synthesized by a number of species from the genus Astragalus (Fabaceae), like A. bisulcatus. An enzyme has been previously isolated from this organism that catalyzes methyl transfer from S-adenosylmethionine to selenocysteine. To elucidate the role of the enzyme in selenium tolerance, the cDNA coding for selenocysteine methyltransferase from A. bisulcatus was cloned and sequenced. Data base searches revealed the existence of several apparent homologs of hitherto unassigned function. The gene for one of them, yagD from Escherichia coli, was cloned, and the protein was overproduced and purified. A functional analysis showed that the YagD protein catalyzes methylation of homocysteine, selenohomocysteine, and selenocysteine with S-adenosylmethionine and S-methylmethionine as methyl group donors. S-Methylmethionine was now shown to be also the physiological methyl group donor for the A. bisulcatus selenocysteine methyltransferase. A model system was set up in E. coli which demonstrated that expression of the plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided that S-methylmethionine is present in the medium. It is postulated that the selenocysteine methyltransferase under selective pressure developed from an S-methylmethionine-dependent thiol/selenol methyltransferase.

Amino Acid Sequence↗

PubMed1999

Acquisition of selenium tolerance by a selenium non-accumulating Astragalus species via selection.

Selection of cultured cells of the selenium sensitive and non-accumulating Astragalus cicer for tolerance to stepwise increasing concentrations of selenite in the medium lead to a variant able to grow at 75 microM selenite. The Se-tolerant culture synthesized a selenocysteine methyltransferase immunologically related but not identical to that of the accumulating A. bisulcatus species and produced Se-methyl-selenocysteine in vivo. Re-cultivation in selenium-free medium lead to breakdown of tolerance and the disappearance of the methyltransferase from cellular proteins. The results prove that the non-accumulating species A. cicer has the cryptic capacity for synthesis of a selenocysteine methyltransferase and also demonstrate that synthesis of the organoselenium compounds in Se-accumulating plants are contributing to selenium tolerance.

Blotting, Western↗

PubMed1999

Genetic probing of the interaction between the translation factor SelB and its mRNA binding element in Escherichia coli.

Decoding of the UGA codon in mRNAs for selenoproteins as selenocysteine requires interaction of the translation factor SelB with an mRNA structure, the SECIS element. A genetic analysis of this interaction was performed by selecting for intergenic suppressor mutations in selB which counteracted the detrimental effect of defined mutations in the SECIS element. Both allele-nonspecific and allele-specific mutations, as judged by readthrough of the UGA into the LacZ-encoding segment of fdhF'-'lacZ fusions and by incorporation of selenium, were isolated. selB genes from ten suppressor mutants were sequenced and the corresponding mutations were localized to five positions within the protein. Four of the suppressors had amino acid exchanges within a 23-amino acid stretch in domain 4b of SelB, which probably represent sites of contact between the protein and the mRNA. A fifth mutation was localized in domain 4a of SelB; it promoted allele-nonspecific readthrough. Since a truncated SelB species lacking domain 4b did not show complex formation with the SECIS element, we speculate that the latter mutation affects the interaction between the tRNA-binding and the mRNA-binding domains. None of the SelB variants was able to promote UGA readthrough when major structural changes that altered the length of the helical part or enlarged the apical loop were introduced into the SECIS element. The results obtained also show that novel pairs of SelB/SECIS derivatives can be generated which may be useful for the targeted insertion of selenocysteine into proteins.

Amino Acid Sequence↗

PubMed1999

Psychosocial factors in children and adolescents with migraine and tension-type headache: a controlled study and review of the literature.

We investigated 341 children and adolescents to evaluate the relevance of psychosocial factors in idiopathic headache. According to the criteria of the International Headache Society, 151 subjects had migraine and 94 had tension-type headache (TTH). Ninety-six subjects were headache-free controls. Psychosocial factors covered family and housing conditions, school problems, relations in the peer group, and several other items. We found that migraine patients did not differ from headache-free controls. Patients with TTH more often had divorced parents than the headache-free controls, and they had fewer peer relations than migraineurs and controls. In addition, migraine patients were significantly more often absent from school due to headache. All other psychosocial factors failed to discriminate between the three study groups. In conclusion, this controlled study in children and adolescents suggests that migraine is not related to family and housing conditions, school situation, or peer relations, whereas TTH is associated with a higher rate of divorced parents and fewer peer relations.

Adaptation, Psychological↗

PubMed1999

Selenocysteine inserting tRNAs: an overview.

One of the recent discoveries in protein biosynthesis was the finding that selenocysteine, the 21st amino acid, is cotranslationally inserted into polypeptides under the direction of a UGA codon assisted by a specific structural signal in the mRNA. The key to selenocysteine biosynthesis and insertion is a special tRNA species, tRNA(Sec). The formation of selenocysteine from serine represents an interesting tRNA-mediated amino acid transformation. tRNA(Sec) (or the gene encoding it) has been found over all three domains of life. It displays a number of unique features that designate it a selenocysteine-inserting tRNA and differentiate it from canonical elongator tRNAs. Although there are still some uncertainties concerning the precise secondary and tertiary structures of eukaryal tRNA(Sec), the major identity determinant for selenocysteine biosynthesis and insertion appears to be the 13 bp long extended acceptor arm. In addition the core of the 3D structure of these tRNAs is different from that of class II tRNAs like tRNA(Sec). The biological implications of these structural differences still remain to be fully understood.

Animals↗

PubMed1999

S-methylmethionine metabolism in Escherichia coli.

Selenium-accumulating Astragalus spp. contain an enzyme which specifically transfers a methyl group from S-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed that Escherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is an S-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants in yagD which also possess defects in metE and metH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine. Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in a metE metH genetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine. Methionine but not S-methylmethionine in the medium reduces the amount of yagD protein. This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon. The physiological roles of the ykfD and yagD products appear to reside in the acquisition of S-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.

Aerobiosis↗

PubMed1998

Substituting selenocysteine for active site cysteine 149 of phosphorylating glyceraldehyde 3-phosphate dehydrogenase reveals a peroxidase activity.

Replacing the essential Cys-149 by a selenocysteine into the active site of phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus leads to a selenoGAPDH that mimics a selenoperoxidase activity. Saturation kinetics were observed with cumenyl and tert-butyl hydroperoxides, with a better catalytic efficiency for the aromatic compound. The enzymatic mechanism fits a sequential model where the formation of a ternary complex between the holoselenoenzyme, the 3-carboxy 4-nitrobenzenethiol used as the reductant and the hydroperoxide precedes product release. The fact that the selenoGAPDH is NAD-saturated supports a binding of hydroperoxide and reductant in the substrate binding site. The catalytic efficiency is similar to selenosubtilisins but remains low compared to selenoglutathione peroxidase. This is discussed in relation to what is known from the X-ray crystal structures of selenoglutathione peroxidase and GAPDHs.

Binding Sites↗

PubMed1998

Bacterial selenocysteine synthase--structural and functional properties.

Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.

Amino Acid Sequence↗

PubMed1998

Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis.

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins. The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix. The tag was fused to the large subunit of [NiFe] hydrogenase 3 (HycE) of Escherichia coli. No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.

Amino Acid Sequence↗

PubMed1998

Selenocysteine inserting RNA elements modulate GTP hydrolysis of elongation factor SelB.

Elongation factor SelB is required for the incorporation of the amino acid selenocysteine into proteins in Escherichia coli. Selenocysteine incorporation is thought to be achieved by simultaneous binding of SelB to selenocysteyl-tRNASec and to an mRNA hairpin structure located 3' adjacent to the UGA selenocysteine codon. SelB was shown previously to bind to GTP or GDP in a molar ratio of 1:1. Here, we demonstrate that SelB, like EF-Tu, exhibits a low intrinsic GTPase activity in the absence of ribosomes. As shown for EF-Tu, GTPase activity of SelB is stimulated by the presence of E. coli 70S ribosomes; the apparent K(m) for GTP hydrolysis is 55 microM. Interestingly, in the presence of the mRNA hairpin which promotes selenocysteine incorporation, GTPase activity of SelB increases additionally by 3-4-fold; stimulation is due to kcat increasing from 0.05/min in the absence to 0.16/min in the presence of the mRNA hairpin. This mRNA-induced stimulation of SelB GTPase activity depends on the presence of ribosomes. The minimal region of the mRNA hairpin capable to stimulate GTP hydrolysis by SelB locates within the upper half of the hairpin; this part of the mRNA structure was demonstrated previously to be sufficient for binding of the mRNA to SelB. On the basis of these results, we propose that binding of the mRNA hairpin to SelB induces a conformational switch within SelB thereby promoting an increase in ribosome-mediated GTP hydrolysis.

Bacterial Proteins↗

PubMed1998

Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase.

The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this self-dependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selDHI) in an E. coli DeltaselD strain. Gene selDHI gave rise to a selenium-containing gene product and also supported - via its activity - the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGASec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif.

Bacterial Proteins↗

PubMed1998

Enzymatic preparation of radiolabeled linear maltodextrins and cyclodextrins of high specific activity from [14C] maltose using amylomaltase, cyclodextrin glucosyltransferase and cyclodextrinase.

Radiolabeled linear and cyclic maltodextrins of high specific radioactivity were prepared using enzymes involved in maltodextrin metabolism. 14C-Labeled maltose was the starting material yielding products of identical specific radioactivity with respect to glucosyl residues. The enzymatic steps involved: i) Formation of linear 14C-labeled maltodexrins (< maltooctaose) using amylomaltase from Escherischia coli; ii) Cyclisation to alpha-cyclodextrin using cyclodextrin-glucosyltransferase of Kiebsiella oxytoca M5a1; iii) Removal of the remaining linear dextrins by amyloglucosidase. The products were purified by paper chromatography, or maltohexaose was specifically obtained from purified alpha-cyclodextrin by the action of cyclodextrinase of K. oxytoca M5a1.

Autoradiography↗

PubMed1998

The periplasmic cyclodextrin binding protein CymE from Klebsiella oxytoca and its role in maltodextrin and cyclodextrin transport.

Klebsiella oxytoca M5a1 has the capacity to transport and to metabolize alpha-, beta- and gamma-cyclodextrins. Cyclodextrin transport is mediated by the products of the cymE, cymF, cymG, cymD, and cymA genes, which are functionally homologous to the malE, malF, malG, malK, and lamB gene products of Escherichia coli. CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding of ligands was analyzed. CymE bound alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin, with dissociation constants (Kd) of 0.02, 0.14 and 0.30 microM, respectively, and linear maltoheptaose, with a Kd of 70 microM. In transport experiments, alpha-cyclodextrin was taken up by the cym system of K. oxytoca three to five times less efficiently than maltohexaose by the E. coli maltose system. Besides alpha-cyclodextrin, maltohexaose was also taken up by the K. oxytoca cym system, but because of the inability of maltodextrins to induce the cym system, growth of E. coli mal mutants on linear maltodextrin was not observed when the cells harbored only the cym uptake system. Strains which gained this capacity by mutation could easily be selected, however.

Bacterial Proteins↗

PubMed1997

SelD homolog from Drosophila lacking selenide-dependent monoselenophosphate synthetase activity.

The isolation and molecular characterization of an invertebrate gene that encodes a homolog of the human selenophosphate synthetase 1 is described. This Drosophila gene, termed selD-like, is located in the cytogenetic interval 50 D/E on the right arm of chromosome 2. It is expressed ubiquitously throughout embryogenesis and found to be highly enriched in the developing gut and in the nervous system of the embryo. The SelD-like from Drosophila was purified after expression in Escherichia coli. The purified protein does not catalyze the selenide-dependent ATP hydrolysis reaction and its gene does not complement a selD lesion in E. coli. These results and the fact that selD-like possesses an arginine residue at the position of the essential Cys17 (E. coli nomenclature) indicate that the Drosophila gene exerts a function different from that of the classical selenophosphate synthetases. Two classes of SelD proteins can therefore be differentiated. The class I proteins contain a cysteine or selenocysteine residue in the active site and display selenide-dependent selenophosphate synthetase activity. Class II proteins, including Drosophila selD-like and human selenophosphate synthetase 1 are devoid of this activity and they possess other amino acids in position 17.

Adenosine Triphosphate↗