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41.
Becker A Rüberg S Baumgarth B Bertram-Drogatz PA Quester I Pühler A 《Journal of molecular microbiology and biotechnology》2002,4(3):187-190
Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes. 相似文献
42.
The consensus concept for thermostability engineering of proteins: further proof of concept 总被引:13,自引:0,他引:13
Lehmann M Loch C Middendorf A Studer D Lassen SF Pasamontes L van Loon AP Wyss M 《Protein engineering》2002,15(5):403-411
Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins. 相似文献
43.
To investigate the influence of the proto-oncogene c-MYC on tumor development in different epithelial tissues which secrete Clara Cell Secretory Protein (uteroglobin, UG), transgenic mouse lines were established expressing the human c-MYC proto-oncogene under the control of the rabbit UG-promoter. These mice expressed the c-MYC transgene in Clara cells and other UG expressing tissues like uterus and prostate. In the bronchioalveolar epithelium of the lung hyperplasias developed originating from Clara cells. Surprisingly, transgenics most frequently developed T-lymphoblastic lymphomas, a polycystic kidney phenotype and renal cell carcinoma derived from tubular epithelial cells, which are both tissues that had so far not been known to express UG. Immunohistological studies in UG/MYC transgenics and in a transgenic line (UG/eGFP) expressing Green Fluorescent Protein confirmed that the uteroglobin promoter is not only active in Clara cells, but also in tubular epithelial cells of the kidney and in lymphatic tissue. The UG/MYC transgenics will be useful to investigate the biochemical mechanisms underlying the development of carcinomas and the oncogenic properties of c-MYC in epithelial cells of various tissues. 相似文献
44.
Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded. 相似文献
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47.
Tannert A Kurz A Erlemann KR Müller K Herrmann A Schiller J Töpfer-Petersen E Manjunath P Müller P 《European biophysics journal : EBJ》2007,36(4-5):461-475
The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine
and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids
and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid
vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts
phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109
to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize
the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that
contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species.
Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented.
Astrid Tannert and Anke Kurz have contributed equally to this work.
Dedicated to Prof. K. Arnold on the occasion of his 65th birthday. 相似文献
48.
Pearce FG Dobson RC Weber A Lane LA McCammon MG Squire MA Perugini MA Jameson GB Robinson CV Gerrard JA 《Biochemistry》2008,47(46):12108-12117
Dihydrodipicolinate synthase (DHDPS) is a tetrameric enzyme that is the first enzyme unique to the ( S)-lysine biosynthetic pathway in plants and bacteria. Previous studies have looked at the important role of Tyr107, an amino acid residue located at the tight-dimer interface between two monomers, in participating in a catalytic triad of residues during catalysis. In this study, we examine the importance of this residue in determining the quaternary structure of the DHDPS enzyme. The Tyr107 residue was mutated to tryptophan, and structural, biophysical, and kinetic studies were carried out on the mutant enzyme. These revealed that while the solid-state structure of the mutant enzyme was largely unchanged, as judged by X-ray crystallography, it exists as a mixture of primarily monomer and tetramer in solution, as determined by analytical ultracentrifugation, size-exclusion chromatography, and mass spectrometry. The catalytic ability of the DHDPS enzyme was reduced by the mutation, which also allowed the adventitious binding of alpha-ketoglutarate to the active site. A reduction in the apparent melting temperature of the mutant enzyme was observed. Thus, the tetrameric quaternary structure of DHDPS is critical to controlling specificity, heat stability, and intrinsic activity. 相似文献
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50.
Enders A Fisch P Schwarz K Duffner U Pannicke U Nikolopoulos E Peters A Orlowska-Volk M Schindler D Friedrich W Selle B Niemeyer C Ehl S 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(8):5060-5068
DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and alphabeta T cells 10-fold, whereas gammadelta T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of gammadelta T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological phenotype. 相似文献