ABSTRACT
During amino-acid crystal fermentation, mechanical stress on bacterial cells caused by crystal collision often impacts negatively on bacterial growth and amino-acid production. When Escherichia coli cells were cultivated under mechanical stress of polyvinyl chloride particles as a model of the crystal fermentation, activities of iron-sulfur (Fe-S) cluster-containing enzymes were apparently decreased. Based on an assumption that function of Fe-S cluster assembly machinery would be elevated to recover the enzyme activities in such stressed cells, we analyzed levels of various components of Fe-S cluster assembly machinery by western blotting. It was found that the expression of HscA, a chaperon component of the machinery, was up-regulated and that shorter forms of HscA with the N-terminal region truncated were accumulated, suggesting an important role of HscA against the mechanical stress. An overexpression of HscA gene in E. coli cells gave a positive effect on rescue of the stress-induced decrease of the activity of Fe-S cluster-containing enzyme. These results may provide a new strategy to alleviate the mechanical stress during the amino-acid crystal fermentation.
Subject(s)
Amino Acids/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , Fermentation , Iron-Sulfur Proteins/metabolism , Stress, Mechanical , Amino Acids/biosynthesis , Amino Acids/chemistry , Batch Cell Culture Techniques , Blotting, Western , Crystallization , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Polyvinyl Chloride , Up-RegulationABSTRACT
To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.
Subject(s)
Ferredoxin-NADP Reductase/chemistry , Thylakoids/enzymology , Zea mays/enzymology , Amino Acid Sequence , Arabidopsis/chemistry , Arabidopsis/enzymology , Arabidopsis/genetics , Base Sequence , Chlorophyll/metabolism , Chloroplasts/enzymology , Crystallization , Electron Transport , Ferredoxin-NADP Reductase/isolation & purification , Ferredoxin-NADP Reductase/metabolism , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Mesophyll Cells/enzymology , Models, Molecular , Molecular Sequence Data , Plant Leaves/chemistry , Plant Leaves/enzymology , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins , Sequence Alignment , Zea mays/chemistry , Zea mays/geneticsABSTRACT
We investigated the mechanical damage to the Escherichia coli cell caused by polyvinyl chloride particles as a model of amino-acid crystal fermentation. Our results indicated that the glucose-consumption rate and the intracellular ATP concentration temporarily increased by the mechanical damage, and decreased after considerable damage had occurred on cell membrane.
Subject(s)
Amino Acids/metabolism , Escherichia coli/metabolism , Fermentation , Adenosine Triphosphate/metabolism , Amino Acids/chemistry , Cell Membrane/drug effects , Cell Membrane/pathology , Escherichia coli/chemistry , Glucose/metabolism , Intracellular Space/chemistry , Polyvinyl Chloride/toxicity , Stress, MechanicalABSTRACT
The light extraction efficiency of top-emitting organic light-emitting diodes (OLEDs) was improved by insertion of a two-dimensional (2D) diffraction layer. The 2D diffraction layer was fabricated by our original nanofabrication technique, the embedded particle monolayer method, which could form a self-assembled particle monolayer. As a result, the electroluminescence intensity of the device with the 2D diffraction layer was improved by 1.67 times (in total luminous flux) and 2.07 times (in peak wavelength). High luminance top-emitting OLEDs were fabricated using the potentially low-cost self-assembling technique.
ABSTRACT
In higher plants, ferredoxin (Fd):NADPH oxidoreductase (FNR) catalyzes reduction of NADP+ in the final step of linear photosynthetic electron transport and is also implicated in cyclic electron flow. We have identified three leaf FNR isoenzymes (LFNR1, LFNR2, and LFNR3) in maize (Zea mays) chloroplasts at approximately equivalent concentrations. Fractionation of chloroplasts showed that, while LFNR3 is an exclusively soluble enzyme, LFNR1 is only found at the thylakoid membrane and LFNR2 has a dual location. LFNR1 and LFNR2 were found to associate with the cytochrome b6f complex following its partial purification. We cloned LFNR3 and produced all three isoenzymes as stable, soluble proteins. Measurement of Fd reduction ability showed no significant differences between these recombinant enzymes. Column chromatography revealed variation between the interaction mechanisms of LFNR1 and LFNR2 with Fd, as detected by differential dependence on specific intermolecular salt bridges and variable sensitivity of interactions to changes in pH. A comparison of LFNR transcripts in leaves of plants grown on variable nitrogen regimes revealed that LFNR1 and LFNR2 transcripts are relatively more abundant under conditions of high demand for NADPH. These results are discussed in terms of the functional differentiation of maize LFNR isoenzymes.
Subject(s)
Chloroplasts/enzymology , Ferredoxin-NADP Reductase/metabolism , Ferredoxins/metabolism , Plant Leaves/enzymology , Zea mays/enzymology , Amino Acid Sequence , Catalysis , Chloroplasts/metabolism , Cloning, Molecular , Cytochrome b6f Complex/metabolism , DNA, Complementary/genetics , Ferredoxin-NADP Reductase/chemistry , Ferredoxin-NADP Reductase/genetics , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Mutation/genetics , Nitrogen/pharmacology , Phylogeny , Plant Leaves/cytology , Plant Leaves/genetics , Plant Leaves/metabolism , Protein Binding , Protein Transport , Quaternary Ammonium Compounds/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thylakoids/metabolism , Zea mays/cytology , Zea mays/genetics , Zea mays/metabolismABSTRACT
Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363-367). Here we report the purification and molecular cloning of endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-beta-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.
