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Purification of QL-nanoKAZ from 800 mL of cultured <i>E</i>. <i>coli</i> cells using a Ni-chelate column.

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Statistics of data collection and structure refinement.

Statistics of data collection and structure refinement.</p

Chloroplast NADPH-Dependent Thioredoxin Reductase from <em>Chlorella vulgaris</em> Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

<div><p>Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in <em>Chlorella vulgaris</em>. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from <em>C. vulgaris</em> and investigated the involvement of <em>Chlorella</em> NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced <em>Chlorella</em> 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature <em>C. vulgaris</em> NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature <em>C. vulgaris</em> 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, <em>tert</em>-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in <em>Chlorella</em>. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of <em>mCvNTRC</em>, and the elevated tolerances were more significant when both <em>mCvNTRC</em> and <em>mCvPrx</em> were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.</p> </div

Luminescence properties of QL-nanoKAZ.

A. Luminescence kinetics of QL-nanoKAZ with CTZ and its analogs as substrates. B. Normalized luminescence spectra of QL-nanoKAZ with CTZ and its analogs, based on the luminescence intensity of QL-nanoKAZ with CTZ. C. Linearity of luminescence intensity (Imax) of QL-nanoKAZ with CTZ, in comparison with nanoKAZ, SNH-nanoKAZ, GLase, and aequorin at the protein concentrations of 0.3 pg to 3 ng (n = 6). Solid and dashed lines represent blank + 3 SD for aequorin and the CTZ-utilizing luciferases, respectively.</p

Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from <i>Gaussia</i> luciferase (GLsp) in CHO-K1 cells.

Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from Gaussia luciferase (GLsp) in CHO-K1 cells.</p

Comparison of the secondary structures between nanoKAZ and QL-nanoKAZ.

The amino acid sequences of nanoKAZ and QL-nanoKAZ are shown with their positions of the secondary structure, and the letters highlighted in orange indicate the substituted 16 amino acid residues in wild KAZ to prepare reverse mutations of nanoKAZ. The cylinders and arrows indicate the regions of α-helices (yellow, α1–α4) and β-strands (blue, β1–β11), respectively. The green in the cylinder (α3) and the arrows (β6 and β7) in QK-nanoKAZ indicate the structural differences compared to nanoKAZ. Tyr 109 is highlighted in red.</p

Primer list used for site-directed mutagenesis to prepare reverse mutant genes for nanoKAZ by PCR.

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Luminescence reaction of coelenterazine (CTZ) catalyzed by the CTZ-utilizing luciferase and chemical structures of CTZ analogs and deaza-CTZ analogs.

A. Oxidation process of CTZ with O2 by CTZ-utilizing luciferases and the degradation product of coelenteramine (CTM), 4-hydroxyphenylacetic acid (4HPAA), and 4-hydroxyphenylpyruvic acid (4HPPA) through 2-peroxycoelenterazine (CTZ-OOH). B. Chemical structures of C2- and C6-modified CTZ analogs. The C6-group of CTZ analogs was colored in red, and the C2- and C8-groups of CTZ analogs were colored in blue. C. Chemical structures of deaza-analogs for CTZ and CTZ-OOH as inhibitors.</p

Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.

Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.</p