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91.
Kiril Tishinov Nikolina Stambolieva Svetla Petrova Boris Galunsky Peter Nedkov 《Acta Physiologiae Plantarum》2009,31(1):199-205
The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at
pH 7.5–8.0 and 45–50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after
the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either α-
or β-position and l-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P1-position and its catalytic efficacy is affected by the structure of both P1 and P1′ parts of the substrate. 相似文献
92.
Microbial metabolism can be harnessed to produce a broad range of industrially important chemicals. Often, three key process variables: Titer, Rate and Yield (TRY) are the target of metabolic engineering efforts to improve microbial hosts toward industrial production. Previous research into improving the TRY metrics have examined the efficacy of having distinct growth and production stages to achieve enhanced productivity. However, these studies assumed a switch from a maximum growth to a maximum production phenotype. Hence, phenotypes with intermediate growth and chemical production in each of the growth and production stages of two-stage processes are yet to be explored. The impact of reduced growth rates on substrate uptake adds to the need for intelligent choice of operating points while designing two-stage processes. In this work, we develop a computational framework that scans the phenotypic space of microbial metabolism to identify ideal growth and production phenotypic targets, to achieve optimal TRY targets. Using this framework, with Escherichia coli as a model organism, we compare two-stage processes that use dynamic pathway regulation, with one-stage processes that use static intervention strategies, for different bioprocess objectives. Our results indicate that two-stage processes with intermediate growth during the production stage always result in optimal TRY values even in cases where substrate uptake is limited due to reduced growth during chemical production. By analyzing the flux distributions for the production enhancing strategies, we identify key reactions and reaction subsystems that require perturbation to achieve a production phenotype for a wide range of metabolites in E. coli. Interestingly, flux perturbations that increase phosphoenolpyruvate and NADPH availability are enriched among these production phenotypes. Furthermore, reactions in the pentose phosphate pathway emerge as key control nodes that function together to increase the availability of precursors to most products in E. coli. The inherently modular nature of microbial metabolism results in common reactions and reaction subsystems that need to be regulated to modify microbes from their target of growth to the production of a diverse range of metabolites. Due to the presence of these common patterns in the flux perturbations, we propose the possibility of a universal production strain. 相似文献
93.
Kim SH Kim SY Park EJ Kim J Park HH So I Kim SJ Jeon JH 《Biochemical and biophysical research communications》2011,415(1):30-35
Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with 31P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by 13C NMR isotopomer analysis of the fate of [U-13C] glucose metabolism.Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media. 相似文献
94.
Yoo A Seo YS Jung JW Sung SK Kim WT Lee W Yang DR 《Journal of structural biology》2006,156(3):407-420
The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe(2+)/H(2)O derived from Petunia hybrida has recently been established by X-ray crystallography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30-15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclusively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the positive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity. 相似文献
95.
Bonifacio A Keizers PH Commandeur JN Vermeulen NP Robert B Gooijer C van der Zwan G 《Biochemical and biophysical research communications》2006,343(3):772-779
Cytochrome P450 2D6 (CYP2D6) is one of the most important drug-metabolizing enzymes in humans. Resonance Raman data, reported for the first time for CYP2D6, show that the CYP2D6 heme is found to be in a six-coordinated low-spin state in the absence of substrates, and it is perturbed to different extents by bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine (MDMA). Dextromethorphan and MDMA induce in CYP2D6 a significant amount of five-coordinated high-spin heme species and reduce the polarity of its heme-pocket, whereas bufuralol does not. Spectra of the F120A mutant CYP2D6 suggest that Phe120 is involved in substrate-binding of dextromethorphan and MDMA, being responsible for the spectral differences observed between these two compounds and bufuralol. These differences could be explained postulating a different substrate mobility for each compound in the CYP2D6 active site, consistently with the role previously suggested for Phe120 in binding dextromethorphan and MDMA. 相似文献
96.
Summary. The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine,
and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic
acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates
by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were
determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl
group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate
affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts
strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated
in this study will be useful for the development of prodrugs. 相似文献
97.
The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics. 相似文献
98.
Jennifer L. Larson-Casey Shubha Murthy Alan J. Ryan A. Brent Carter 《The Journal of biological chemistry》2014,289(52):36204-36219
Protein kinase B (Akt) is a key effector of multiple cellular processes, including cell survival. Akt, a serine/threonine kinase, is known to increase cell survival by regulation of the intrinsic pathway for apoptosis. In this study, we found that Akt modulated the mevalonate pathway, which is also linked to cell survival, by increasing Rho GTPase activation. Akt modulated the pathway by phosphorylating mevalonate diphosphate decarboxylase (MDD) at Ser96. This phosphorylation in macrophages increased activation of Rac1, which enhanced macrophage survival because mutation of MDD (MDDS96A) induced apoptosis. Akt-mediated activation in macrophages was specific for Rac1 because Akt did not increase activity of other Rho GTP-binding proteins. The relationship between Akt and Rac1 was biologically relevant because Akt+/− mice had significantly less active Rac1 in alveolar macrophages, and macrophages from Akt+/− mice had an increase in active caspase-9 and -3. More importantly, Akt+/− mice were significantly protected from the development of pulmonary fibrosis, suggesting that macrophage survival is associated with the fibrotic phenotype. These observations for the first time suggest that Akt plays a critical role in the development and progression of pulmonary fibrosis by enhancing macrophage survival via modulation of the mevalonate pathway. 相似文献
99.
Takashi Muranaka 《Landscape and Ecological Engineering》2009,5(1):11-21
Aster kantoensis, an endangered and monocarpic perennial plant species, is endemic to the gravelly floodplain of a few rapid flowing rivers
in eastern central Japan. In recent years, an extreme declining trend in the species has been accelerated due to the strong
negative influence by invasion of an alien grass, Eragrostis curvula. A restoration project aimed at recovering the original condition of the floodplain in the Kinu River, central Japan, has
been started. To determine the possibility of successful restoration as well as its habitat preferences, I carried out some
seed sowing experiments. In April 2003, seeds collected from a seminatural habitat were sown (54,000 seeds) in the restoration
site (1.2 ha), where flood frequency, substrate condition, and control of alien plants are combined to form different habitat
conditions. Seedling survival, flowery, and seed production were subsequently monitored from 2003 to 2005. Seed cohorts completed
their life cycles within 3 years, and mean fitness of 927 was achieved. Performance of A. kantoensis seedlings was generally greater for environmental variables of sandy-type substrate and/or with control of alien plants.
In addition, there were significant negative correlations between percentage survival, percentage flowery, and seed production
with vegetation cover and coverage of E. curvula. The results confirm that, if safe sites with sparse vegetation exist, irrespective of their substrate condition, as well
as seed sources of river endemics in natural habitats, restoration of riparian vegetation including river endemics is possible.
The aggressive alien species E. curvula should be taken into consideration. 相似文献
100.
Microbial 2,3-butanediol production: a state-of-the-art review 总被引:7,自引:0,他引:7
2,3-Butanediol is a promising bulk chemical due to its extensive industry applications. The state-of-the-art nature of microbial 2,3-butanediol production is reviewed in this paper. Various strategies for efficient and economical microbial 2,3-butanediol production, including strain improvement, substrate alternation, and process development, are reviewed and compared with regard to their pros and cons. This review also summarizes value added derivatives of biologically produced 2,3-butanediol and different strategies for downstream processing. The future prospects of microbial 2,3-butanediol production are discussed in light of the current progress, challenges, and trends in this field. Guidelines for future studies are also proposed. 相似文献