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Mingxuan Wang Jing Li Wenjie Cong Jianguo Zhang 《Current issues in molecular biology》2022,44(5):1828
Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min−1 mg−1 and 10.0 ± 0.5 nmol min−1 mg−1, respectively. The highest kcat/Km value of ω3 and ω6 desaturase was 0.41 µM−1 min−1 and 0.09 µM−1 min−1 when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina. 相似文献
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Role of oxygen supply in α, ω‐dodecanedioic acid biosynthesis from n‐dodecane by Candida viswanathii ipe‐1: Effect of stirring speed and aeration
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Weifeng Cao Yujue Wang Jianquan Luo Junxiang Yin Yinhua Wan 《Engineering in Life Science》2018,18(3):196-203
α, ω‐Dodecanedioic acid (DC12) usually serves as a monomer of polyamides or some special nylons. During the biosynthesis, oxygenation cascaded in conversion of hydrophobic n‐dodecane to DC12, while the oxidation of n‐dodecane took place in the intracellular space. Therefore, it was important to investigate the role of oxygen supply on the cell growth and DC12 biosynthesis. It was found that stirring speed and aeration influenced the dissolved oxygen (DO) concentration which in turn affected cell growth as well as DC12 biosynthesis. However, the effect of culture redox potential (Orp) level on DC12 biosynthesis was more significant than that of DO level. For DC12 biosynthesis, the first step was to form the emulsion droplets through the interaction of n‐dodecane and the cell. When the stirring speed was enhanced, slits in the surface layer of the emulsion droplets would be increased. Thus, the substances transportation by water through the slits would be intensified, leading to an enhanced DC12 production. Compared with the batch culture at a lower stirring speed (400 rpm) without culture redox potential (Orp) control, the DC12 concentration was increased by 5 times up to 201.3 g/L with Orp controlled above 0 mV at a higher stirring speed (800 rpm). 相似文献
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Joo‐Hyun Seo Dohyun Kyung Keehyoung Joo Jooyoung Lee Byung‐Gee Kim 《Biotechnology and bioengineering》2011,108(2):253-263
The half reactions of ω‐aminotransferase (ω‐AT) from Vibrio fluvialis JS17 (ω‐ATVf) were carried out using purified pyridoxal 5′‐phosphate‐enzyme (PLP‐Enz) and pyridoxamine 5′‐phosphate‐enzyme (PMP‐Enz) complexes to investigate the relative activities of substrates. In the reaction generating PMP‐Enz from PLP‐Enz using L ‐alanine as an amine donor, L ‐alanine showed about 70% of the initial reaction rate of (S)‐α‐methylbenzylamine ((S)‐α‐MBA). However, in the subsequent half reaction recycling PLP‐Enz from PMP‐Enz using acetophenone as an amine acceptor, acetophenone showed nearly negligible reactivity compared to pyruvate. These results indicate that the main bottleneck in the asymmetric synthesis of (S)‐α‐MBA lies not in the amination of PLP by alanine, but in the amination of acetophenone by PMP‐Enz, where conformational restraints of the enzyme structure is likely to be the main reason for limiting the amine group transfer from PMP‐Enz to acetophenone. Based upon those half reaction experiments using the two amino acceptors of different activity, it appears that the relative activities of the two amine donors and the two acceptors involved in the ω‐AT reactions can roughly determine the asymmetric synthesis yield of the target chiral amine compound. Predicted conversion yields of several target chiral amines were calculated and compared with the experimental conversion yields. Approximately, a positive linear correlation (Pearson's correlation coefficient = 0.92) was observed between the calculated values and the experimental conversion yields. To overcome the low (S)‐α‐MBA productivity of ω‐ATVf caused by the possible disadvantageous structural constraints for acetophenone, new ω‐ATs showing higher affinity to benzene ring of acetophenone than ω‐ATVf were computationally screened using comparative modeling and protein‐ligand docking. ω‐ATs from Streptomyces avermitilis MA‐4680 (SAV2612) and Agrobacterium tumefaciens str. C58 (Atu4761) were selected, and the two screened ω‐ATs showed higher asymmetric synthesis reaction rate of (S)‐α‐MBA and lower (S)‐α‐MBA degradation reaction rate than ω‐ATVf. To verify the higher conversion yield of the variants of ω‐ATs, the reaction with 50 mM acetophenone and 50 mM alanine was performed with coupling of lactate dehydrogenase and two‐phase reaction system. SAV2612 and Atu4761 showed 70% and 59% enhanced yield in the synthesis of (S)‐α‐MBA compared to that of ω‐ATVf, respectively. Biotechnol. Bioeng. 2011;108: 253–263. © 2010 Wiley Periodicals, Inc. 相似文献
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Our purpose was to identify the sequence of ω-amidase, which hydrolyses the amide group of α-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative ‘carbon-nitrogen hydrolase’. The closest mammalian homolog of this putative bacterial ω-amidase is ‘nitrilase 2’, whose size and amino acid composition were in good agreement with those reported for purified rat liver ω-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of α-ketoglutaramate and other known substrates of ω-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is ω-amidase. 相似文献
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Georgios A. Dalkas Fabian Teheux Jean Marc Kwasigroch Marianne Rooman 《Proteins》2014,82(9):1734-1746
The identification of immunogenic regions on the surface of antigens, which are able to stimulate an immune response, is a major challenge for the design of new vaccines. Computational immunology aims at predicting such regions—in particular B‐cell epitopes—but is far from being reliably applicable on a large scale. To gain understanding into the factors that contribute to the antigen–antibody affinity and specificity, we perform a detailed analysis of the amino acid composition and secondary structure of antigen and antibody surfaces, and of the interactions that stabilize the complexes, in comparison with the composition and interactions observed in other heterodimeric protein interfaces. We make a distinction between linear and conformational B‐cell epitopes, according to whether they consist of successive residues along the polypeptide chain or not. The antigen–antibody interfaces were shown to differ from other protein–protein interfaces by their smaller size, their secondary structure with less helices and more loops, and the interactions that stabilize them: more H‐bond, cation–π, amino–π, and π–π interactions, and less hydrophobic packing; linear and conformational epitopes can clearly be distinguished. Often, chains of successive interactions, called cation/amino–π and π–π chains, are formed. The amino acid composition differs significantly between the interfaces: antigen–antibody interfaces are less aliphatic and more charged, polar and aromatic than other heterodimeric protein interfaces. Moreover, paratopes and epitopes—albeit to a lesser extent—have amino acid compositions that are distinct from general protein surfaces. This specificity holds promise for improving B‐cell epitope prediction. Proteins 2014; 82:1734–1746. © 2014 Wiley Periodicals, Inc. 相似文献
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Boris F. Krasnikov Chin-Hsiang Chien Regina Nostramo John T. Pinto Edward Nieves Myrasol Callaway Jin Sun Kay Huebner Arthur J.L. Cooper 《Biochimie》2009,91(9):1072-1080
The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is ω-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated ω-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are α-ketoglutaramate and α-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of α-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating α-keto-γ-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess α-ketoglutaramate may be neurotoxic. Moreover, α-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of ω-amidase is to remove potentially toxic intermediates by converting α-ketoglutaramate and α-ketosuccinamate to biologically useful α-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of ω-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology. 相似文献
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ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines
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Joungjwa Ahn Youn Wook Chung Jin‐Byung Park Kyung Mi Yang 《Journal of cellular biochemistry》2018,119(1):998-1007
ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth. 相似文献
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M. Berres 《Biometrical journal. Biometrische Zeitschrift》1983,25(6):527-535
Computations have been performed to find an adequate definition of exact two-sided probabilities in 2times2 contingency tables. It turns out, that both uncorrected χ2 and Yates' correction for continuity give only unsatisfactory approximations to the exact probabilities of the hypergeometric distribution. The latter are therefore recommended for general use. 相似文献
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Knockdown or inhibition of SIRT2 enhances biological stress-tolerance. We extend this phenotype showing that SIRT2 knockdown reduces anoxia-reoxygenation injury in H9c2 cells. Gene array analysis following SIRT2 siRNA knockdown identifies 14-3-3 zeta as the most robustly induced gene. SIRT2 knockdown evokes induction of this chaperone, facilitating cytosolic sequestration of BAD with a corresponding reduction in mitochondrial BAD localization. Concurrent siRNA against SIRT2 and 14-3-3 zeta abolishes the SIRT2-depleted cytoprotective phenotype. SIRT2 functions to moderate cellular stress-tolerance, in part, by modulating the levels of 14-3-3 zeta with the concordant control of BAD subcellular localization. 相似文献
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Christian Wiese Eva Große Maestrup Dirk Schepmann Stefan Grimme Hans‐Ulrich Humpf Peter Brust Bernhard Wünsch 《Chirality》2011,23(2):148-154
It was shown that racemic (±)‐ 2 [1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine], WMS‐1813 ] represents a promising positron emission tomography (PET) tracer for the investigation of centrally located σ1 receptors. To study the pharmacological activity of the enantiomers of 2 , a preparative HPLC separation of (R)‐2 and (S)‐2 was performed. The absolute configuration of the enantiomers was determined by CD‐spectroscopy together with theoretical calculations of the CD‐spectrum of a model compound. In receptor binding studies with the radioligand [3H]‐(+)‐pentazocine, (S)‐2 was thrice more potent than its (R)‐configured enantiomer (R)‐2 . The metabolic degradation of the more potent (S)‐enantiomer was considerably slower than the metabolism of (R)‐2 . The structures of the main metabolites of both enantiomers were elucidated by determination of the exact mass using an Orbitrap‐LC‐MS system. These experiments showed a stereoselective biotransformation of the enantiomers of 2 . Chirality, 2011. © 2010 Wiley‐Liss, Inc. 相似文献
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Dunming Zhu Brooke A. Hyatt Ling Hua 《Journal of Molecular Catalysis .B, Enzymatic》2009,56(4):272-276
An alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (PFADH) effectively catalyzed the reductions of various substituted α-chloroacetophenones to furnish the corresponding (R)-configurated α-chlorohydrins with excellent enantiomeric purity. The co-factor NADH could be recycled with d-glucose dehydrogenase/d-glucose system or in a coupled substrate approach using iso-propanol as the hydrogen donor. The hydrogen transfer mode should be more cost-effective. Thus, the PFADH-catalyzed hydrogen transfer reductions of some substrates were carried out on the preparative scale, demonstrating that this enzyme would be a valuable biocatalyst for the preparation of chiral chlorohydrins of pharmaceutical interest. 相似文献
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Cytotoxic (9βH)‐Pimarane and (9βH)‐17‐Norpimarane Diterpenes from the Tuber of Icacina trichantha
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Michael Monday Onakpa Ming Zhao Tanja Gödecke Wei‐Lun Chen Chun‐Tao Che Bernard D. Santarsiero Steven M. Swanson Isaac Uzoma Asuzu 《化学与生物多样性》2014,11(12):1914-1922
Three (9βH)‐pimaranes, 1, 2 , and 3 , and two (9βH)‐17‐norpimaranes, 4 and 5 , belonging to a rare compound class in nature, were obtained from the tubers of Icacina trichantha for the first time. Compound 1 is a new natural product, and 2 – 5 have been previously reported. The structures were elucidated based on NMR and MS data, and optical rotation values. The absolute configurations of (9βH)‐pimaranes were unambiguously established based on X‐ray crystallographic analysis. Full NMR signal assignments for the known compounds 2, 4 , and 5 , which were not available in previous publications, are also reported. All five isolates displayed cytotoxic activities on MDA‐MB‐435 cells (IC50 0.66–6.44 μM ), while 2, 3 , and 4 also exhibited cytotoxicities on HT‐29 cells (IC50 3.00–4.94 μM ). 相似文献
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M. Schemper 《Biometrical journal. Biometrische Zeitschrift》1987,29(8):1003-1009
Several methods of testing general one-sample and two-sample hypotheses of Kendall's τ are discussed. The performance of these procedures in typical situations likely to occur in practice was investigated by numerous Monte Carlo experiments. The common one-sample test (H0: τ = 0) based on the permutational variance performs very similar to bootstrap or jackknife testing for true τ near zero. For true |τ| perhaps greater than 0.4 and moderate sample sizes bootstrap techniques are the methods of choice. 相似文献
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The family of 14‐3‐3 proteins and specifically 14‐3‐3σ are up‐regulated during the development of renal pathologies
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Myrto Rizou Eleni A. Frangou Filio Marineli Niki Prakoura Jerome Zoidakis Harikleia Gakiopoulou George Liapis Panagiotis Kavvadas Christos Chatziantoniou Manousos Makridakis Antonia Vlahou John Boletis Demetrios Vlahakos Dimitrios Goumenos Evgenios Daphnis Christos Iatrou Aristidis S. Charonis 《Journal of cellular and molecular medicine》2018,22(9):4139-4149