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21.
Brison Y Pijning T Malbert Y Fabre É Mourey L Morel S Potocki-Véronèse G Monsan P Tranier S Remaud-Siméon M Dijkstra BW 《The Journal of biological chemistry》2012,287(11):7915-7924
ΔN123-glucan-binding domain-catalytic domain 2 (ΔN123-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN123-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN123-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN123-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN123-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches. 相似文献
22.
Thorsten Eggert Christian Leggewie Michael Puls Wolfgang Streit Gertie van Pouderoyen Bauke W. Dijkstra Karl-erich Jaeger 《Biocatalysis and Biotransformation》2004,22(2):141-146
Enzymes produced from bacteria and eukaryotic organisms are presently being used for a large variety of different biotechnological applications. The rapidly increasing demand for enzymes which are active towards novel and often non-natural substrates has triggered the development of novel molecular biological methods of enzyme isolation and design. The metagenome approach is a cultivation-independent method which allows the direct cloning and expression of environmental DNA thereby providing access to a wealth of so-far unknown biocatalysts. Additionally, newly identified or existing biocatalysts can be further optimized by different methods of directed evolution. Here, the principle of the metagenome approach is outlined and a strategy is presented for the optimization of a bacterial lipase using a combination of rational design and directed evolution. 相似文献
23.
The genetic organization of the determinants of type 1, K88ab, K99 fimbriae and P(pap)pili of Escherichia coli is presented. The functions of the various gene products are described and a model for the process of fimbriae biogenesis is presented and discussed. 相似文献
24.
P A Scotti Q A Valent E H Manting M L Urbanus A J Driessen B Oudega J Luirink 《The Journal of biological chemistry》1999,274(42):29883-29888
In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes. 相似文献
25.
Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a
decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were
located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type
1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected
type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting
LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene
surface could be observed.
Received: 1 December 1998 / Accepted: 3 April 1999 相似文献
26.
27.
Bauke Schievink Hiddo Lambers Heerspink Hubert Leufkens Dick De Zeeuw Jarno Hoekman 《PloS one》2014,9(9)
Aim
There is discussion whether medicines can be authorized on the market based on evidence from surrogate endpoints. We assessed opinions of different stakeholders on this topic.Methods
We conducted an online questionnaire that targeted various stakeholder groups (regulatory agencies, pharmaceutical industry, academia, relevant public sector organisations) and medical specialties (cardiology or nephrology vs. other). Participants were enrolled through purposeful sampling. We inquired for conditions under which surrogate endpoints can be used, the validity of various cardio-renal biomarkers and new approaches for biomarker use.Results
Participants agreed that surrogate endpoints can be used when the surrogate is scientifically valid (5-point Likert response format, mean score: 4.3, SD: 0.9) or when there is an unmet clinical need (mean score: 3.8, SD: 1.2). Industry participants agreed to a greater extent than regulators and academics. However, out of four proposed surrogates (blood pressure (BP), HbA1c, albuminuria, CRP) for cardiovascular outcomes or end-stage renal disease, only use of BP for cardiovascular outcomes was deemed moderately accurate (mean: 3.6, SD: 1.1). Specialists in cardiology or nephrology tended to be more positive about the use of surrogate endpoints.Conclusion
Stakeholders in drug development do not oppose to the use of surrogate endpoints in drug marketing authorization, but most surrogates are not considered valid. To solve this impasse, increased efforts are required to validate surrogate endpoints and to explore alternative ways to use them. 相似文献28.
An improved neutral landscape model for recreating real landscapes and generating landscape series for spatial ecological simulations 总被引:1,自引:0,他引:1 下载免费PDF全文
Maarten J. van Strien Cornelis T. J. Slager Bauke de Vries Adrienne Grêt‐Regamey 《Ecology and evolution》2016,6(11):3808-3821
Many studies have assessed the effect of landscape patterns on spatial ecological processes by simulating these processes in computer‐generated landscapes with varying composition and configuration. To generate such landscapes, various neutral landscape models have been developed. However, the limited set of landscape‐level pattern variables included in these models is often inadequate to generate landscapes that reflect real landscapes. In order to achieve more flexibility and variability in the generated landscapes patterns, a more complete set of class‐ and patch‐level pattern variables should be implemented in these models. These enhancements have been implemented in Landscape Generator (LG), which is a software that uses optimization algorithms to generate landscapes that match user‐defined target values. Developed for participatory spatial planning at small scale, we enhanced the usability of LG and demonstrated how it can be used for larger scale ecological studies. First, we used LG to recreate landscape patterns from a real landscape (i.e., a mountainous region in Switzerland). Second, we generated landscape series with incrementally changing pattern variables, which could be used in ecological simulation studies. We found that LG was able to recreate landscape patterns that approximate those of real landscapes. Furthermore, we successfully generated landscape series that would not have been possible with traditional neutral landscape models. LG is a promising novel approach for generating neutral landscapes and enables testing of new hypotheses regarding the influence of landscape patterns on ecological processes. LG is freely available online. 相似文献
29.
Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13. 总被引:8,自引:4,他引:8 下载免费PDF全文
B Oudega F Stegehuis G J van Tiel-Menkveld F K de Graaf 《Journal of bacteriology》1982,150(3):1115-1121
Excretion of cloacin DF13 was studied in Escherichia coli cells harboring different CloDF13 insertion and deletion mutant plasmids. Insertions of a transposon at position 9.8 or 11.5% of the CloDF13 plasmid blocked the expression of gene H and strongly reduced the specific excretion of cloacin DF13 into the culture medium, but had no effect on the production of cloacin DF13. Insertions in or deletions of regions of the CloDF13 DNA upstream the cloacin operon did not affect the excretion or production of the bacteriocin. Introduction of a CloDF13 plasmid that encodes for the gene H product in cells harboring a CloDF13 plasmid with an insertion in gene H stimulated the excretion of cloacin DF13 significantly in mitomycin C-induced and in noninduced cultures. Cloacin DF13 in cloacinogenic cells that did not produce the gene H protein was found to be about 90% located in the cytoplasm. In cells that did produce the gene H product, about 30% of the cloacin DF13 molecules were found in the cytoplasm, about 18% were found in the periplasm, about 2% were in the membranes, and about 50% were located in the culture supernatant. Cyclic AMP stimulated the production but not the excretion of cloacin DF13 in cells cultivated in the presence of glucose. 相似文献
30.
Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity 下载免费PDF全文
Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup. Its 1.8 A crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer. The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites. Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone-effector complexes are structurally conserved and function as three-dimensional secretion signals. 相似文献