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L-Lactate cytochrome c oxidoreductase (flavocytochrome b 2, FC b 2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b 2 producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b 2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b 2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b 2 producer characterized by a sixfold increased (to 3 μmol min?1 mg?1 protein in cell-free extract) activity of the enzyme. 相似文献
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Birte Plitzko Gudrun Ott Debora Reichmann Colin J. Henderson C. Roland Wolf Ralf Mendel Florian Bittner Bernd Clement Antje Havemeyer 《The Journal of biological chemistry》2013,288(28):20228-20237
The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(11):3063-3066
Using a DNA microarray, we found that expression of the genes related to lactate metabolism was upregulated in a lactate-producing recombinant Saccharomyces cerevisiae strain. Disruption of the CYB2 gene encoding L-lactate dehydrogenase improved the L-lactate production by S. cerevisiae under low pH condition. 相似文献
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Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxy resins, has previously been reported to induce micronuclei containing whole chromosomes in Chinese hamster V79 cells. In the present study, the aneuploidogenic potential of BPA was investigated in cultured human AG01522C fibroblasts. In contrast to the known aneugens diethylstilbestrol (DES) and 17beta-estradiol, which caused mitotic arrest and the induction of kinetochore-positive micronuclei, BPA did not induce micronuclei and inhibited the proliferation of AG01522C cells in G2 phase and probably also in G1 phase. Fluorescence microscopy of the BPA-treated cells after immunofluorescent staining of microtubules revealed structural abnormalities of the cytoplasmic microtubule complex (CMTC): densely stained rings and loops of tubulin were observed, which increased in number with increasing BPA concentration and were more stable against low temperature than normal microtubules. The mechanisms of the growth inhibition and the interference with microtubules elicited by BPA in AG01522C cells are presently unknown. The formation of rings and loops in the CMTC of AG01522C cells was also observed with two congeners of BPA carrying one and two, respectively, additional methyl groups in ortho-position to the phenolic hydroxyl group at each aromatic ring. However, in contrast to BPA itself, these congeners of BPA behaved "DES-like" by inducing mitotic arrest and kinetochore-positive micronuclei in AG01522C cells. 相似文献
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Overexpression of CYB5R3 and NQO1, two NAD+‐producing enzymes,mimics aspects of caloric restriction 下载免费PDF全文
Alberto Diaz‐Ruiz Michael Lanasa Joseph Garcia Hector Mora Frances Fan Alejandro Martin‐Montalvo Andrea Di Francesco Miguel Calvo‐Rubio Andrea Salvador‐Pascual Miguel A. Aon Kenneth W. Fishbein Kevin J. Pearson Jose Manuel Villalba Placido Navas Michel Bernier Rafael de Cabo 《Aging cell》2018,17(4)
Calorie restriction (CR) is one of the most robust means to improve health and survival in model organisms. CR imposes a metabolic program that leads to increased stress resistance and delayed onset of chronic diseases, including cancer. In rodents, CR induces the upregulation of two NADH‐dehydrogenases, namely NAD(P)H:quinone oxidoreductase 1 (Nqo1) and cytochrome b5 reductase 3 (Cyb5r3), which provide electrons for energy metabolism. It has been proposed that this upregulation may be responsible for some of the beneficial effects of CR, and defects in their activity are linked to aging and several age‐associated diseases. However, it is unclear whether changes in metabolic homeostasis solely through upregulation of these NADH‐dehydrogenases have a positive impact on health and survival. We generated a mouse that overexpresses both metabolic enzymes leading to phenotypes that resemble aspects of CR including a modest increase in lifespan, greater physical performance, a decrease in chronic inflammation, and, importantly, protection against carcinogenesis, one of the main hallmarks of CR. Furthermore, these animals showed an enhancement of metabolic flexibility and a significant upregulation of the NAD+/sirtuin pathway. The results highlight the importance of these NAD+ producers for the promotion of health and extended lifespan. 相似文献
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The highly invasive and chemoresistant phenotype of pancreatic cancer highlights the urgency to identify prognostic biomarkers and novel therapeutic targets. Recently, we observed a significant correlation between shorter survival and loss of the cytoband 18q22.3. Here we investigated genes encoded by this cytoband, and demonstrated the prognostic value of CYB5A in resected and metastatic patients. Furthermore, our in vitro and in vivo studies clarified CYB5A inhibitory activity of oncogenic phenotypes through autophagy induction. This raises the possibility that inhibition of CYB5A-deregulated downstream pathways, such as those involving TRAF6, may favor autophagy-mediated cancer cell death in selected subgroups of patients. 相似文献
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Cytochrome bc1 is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson-Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc1. Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc1 from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Qo-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron-sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Qo-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Qi-site) is supported by our results. A modified path for proton uptake towards the Qi-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c1 subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Qi-site stabilises protons in this cluster of lysine residues. 相似文献
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