Publications by authors named "Yoshihiro Sawa"

45 Publications

The D-mannose/L-galactose pathway is the dominant ascorbate biosynthetic route in the moss Physicomitrium patens.

Plant J 2021 Jul 10. Epub 2021 Jul 10.

Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the D-mannose/L-galactose (D-Man/L-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophyte, while the D-galacturonate (D-GalUA) pathway is distributed in parasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the D-Man/L-Gal pathway and an orthologous gene encoding aldonolactonae (ALase) - a key enzyme for the D-GalUA pathway - Physicomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. P. patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of L-galactonic acid to the final precursor L-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encodes the rate-limiting enzyme GDP-L-Gal phosphorylase in the D-Man/L-Gal pathway, markedly was decreased to 46% and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthestic pathway in P.patens is the D-Man/L-Gal pathway, not the D-GalUA pathway and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.
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http://dx.doi.org/10.1111/tpj.15413DOI Listing
July 2021

Cooperation of chloroplast ascorbate peroxidases and proton gradient regulation 5 is critical for protecting Arabidopsis plants from photo-oxidative stress.

Plant J 2021 May 24. Epub 2021 May 24.

Graduate School of Natural Science and Technology, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

High-light (HL) stress enhances the production of H O from the photosynthetic electron transport chain in chloroplasts, potentially causing photo-oxidative damage. Although stromal and thylakoid membrane-bound ascorbate peroxidases (sAPX and tAPX, respectively) are major H O -scavenging enzymes in chloroplasts, their knockout mutants do not exhibit a visible phenotype under HL stress. Trans-thylakoid proton gradient (∆pH)-dependent mechanisms exist for controlling H O production from photosynthesis, such as thermal dissipation of light energy and downregulation of electron transfer between photosystems II and I, and these may compensate for the lack of APXs. To test this hypothesis, we focused on a proton gradient regulation 5 (pgr5) mutant, wherein both ∆pH-dependent mechanisms are impaired, and an Arabidopsis sapx tapx double mutant was crossed with the pgr5 single mutant. The sapx tapx pgr5 triple mutant exhibited extreme sensitivity to HL compared with its parental lines. This phenotype was consistent with cellular redox perturbations and enhanced expression of many oxidative stress-responsive genes. These findings demonstrate that the PGR5-dependent mechanisms compensate for chloroplast APXs, and vice versa. An intriguing finding was that the failure of induction of non-photochemical quenching in pgr5 (because of the limitation in ∆pH formation) was partially recovered in sapx tapx pgr5. Further genetic studies suggested that this recovery was dependent on the NADH dehydrogenase-like complex-dependent pathway for cyclic electron flow around photosystem I. Together with data from the sapx tapx npq4 mutant, we discuss the interrelationship between APXs and ∆pH-dependent mechanisms under HL stress.
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http://dx.doi.org/10.1111/tpj.15352DOI Listing
May 2021

Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light-Induced Ascorbate Accumulation.

Plant Physiol 2020 05 23;183(1):112-122. Epub 2020 Mar 23.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Shimane 690-8504, Japan

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis () triple mutant lacking all three genes (herein called ∆) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated ∆ Arabidopsis plants as well as a quadruple mutant line combining ∆ with an additional mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the mutant that contains ∼30% of the wild-type GSH level. The mutant accumulated ascorbate at a wild-type level under high light; however, when the mutation was combined with ∆, there was near-complete inhibition of high-light-dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in ∆ plants, and that both DHAR activity and GSH content set a threshold for high-light-induced ascorbate accumulation.
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http://dx.doi.org/10.1104/pp.19.01556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210653PMC
May 2020

Comparative proteomic analysis of mitochondria isolated from Euglena gracilis under aerobic and hypoxic conditions.

PLoS One 2019 31;14(12):e0227226. Epub 2019 Dec 31.

Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, Matsue, Shimane, Japan.

The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, suggesting contribution of post-transcriptional regulation. In this study, we conducted a proteome analysis of E. gracilis mitochondria, as this organelle employs the fatty-acid synthesis pathway under hypoxic conditions. Mitochondria were isolated from E. gracilis SM-ZK strain treated with both aerobic and hypoxic conditions and used for shotgun proteomic analysis. Three independent proteomic analyses succeeded in identifying a total of 714 non-redundant proteins. Of these, 229 were detected in common to all experiments, and 116 were significantly recognized as differentially expressed proteins. GO enrichment analysis suggested dynamic changes in mitochondrial metabolic pathways and redox reactions under aerobic and hypoxic conditions. Protein levels of bifunctional enzymes isocitrate lyase and malate synthase in glyoxylate cycle were 1.35-fold higher under hypoxic conditions. Abundances of the propionyl-CoA synthetic enzymes, succinyl-CoA synthetase and propionyl-CoA carboxylase, were also 1.35- and 1.47-fold higher, respectively, under hypoxic conditions. Protein levels of pyruvate:NADP+ oxidoreductase, a key enzyme for anaerobic synthesis of acetyl-CoA, which serves as a C2 donor for fatty acids, showed a 1.68-fold increase under hypoxic conditions, whereas those of pyruvate dehydrogenase subunits showed a 0.77-0.81-fold decrease. Protein levels of the fatty-acid synthesis enzymes, 3-ketoacyl-CoA thiolase isoforms (KAT1 and KAT2), 3-hydroxyacyl-CoA dehydrogenases, and acyl-CoA dehydrogenase were up-regulated by 1.20- to 1.42-fold in response to hypoxic treatment. Overall, our proteomic analysis revealed that wax ester synthesis-related enzymes are up-regulated at the protein level post-transcriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938325PMC
April 2020

Chloroplast development activates the expression of ascorbate biosynthesis-associated genes in Arabidopsis roots.

Plant Sci 2019 Jul 16;284:185-191. Epub 2019 Apr 16.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan; Graduate School of Natural Science and Technology, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan; Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

Transcriptional activation of ascorbate biosynthesis-associated genes under illumination is one of the important steps in ascorbate pool size regulation in photosynthetic tissues. Several biological processes within chloroplasts such as photosynthesis are required for this activation, suggesting functional chloroplasts to play a key role. We herein found that when grown on agar plate, ascorbate content in Arabidopsis non-photosynthetic tissues, roots, are unexpectedly almost comparable to that in shoots. The high accumulation of ascorbate was particularly observed in root regions closer to the root-hypocotyl junction, in which chloroplast development occurred because of a direct exposure to light. When chloroplast development in roots were further stimulated by shoot removal, the expression of biosynthetic genes, especially VTC2 gene that encodes GDP-l-galactose phosphorylase, was activated, resulting in an increase in ascorbate pool size. These positive effects were canceled when the roots were treated with a photosynthetic inhibitor. A null mutation in the LONG HYPOCOTYL 5 (HY5) gene almost completely inhibited root greening as well as the VTC2 expression. Overall, these findings show that chloroplast development can trigger the expression of ascorbate biosynthesis-associated genes not only in leaves but also in roots.
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http://dx.doi.org/10.1016/j.plantsci.2019.04.012DOI Listing
July 2019

A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

PLoS One 2019 16;14(1):e0210755. Epub 2019 Jan 16.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334954PMC
November 2019

Affinity resins as new tools for identifying target proteins of ascorbic acid.

Analyst 2018 Feb;143(4):874-882

Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023, Japan.

l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.
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http://dx.doi.org/10.1039/c7an01592eDOI Listing
February 2018

Prediction of arsenic and antimony transporter major intrinsic proteins from the genomes of crop plants.

Int J Biol Macromol 2018 Feb 26;107(Pt B):2630-2642. Epub 2017 Oct 26.

Department of Life Science and Biotechnology, Shimane University, Shimane 690-8504, Japan.

Major intrinsic proteins (MIPs), commonly known as aquaporins, transport water and non-polar small solutes. Comparing the 3D models and the primary selectivity-related motifs (two Asn-Pro-Ala (NPA) regions, the aromatic/arginine (ar/R) selectivity filter, and Froger's positions (FPs)) of all plant MIPs that have been experimentally proven to transport arsenic (As) and antimony (Sb), some substrate-specific signature sequences (SSSS) or specificity determining sites (SDPs) have been predicted. These SSSS or SDPs were determined in 543 MIPs found in the genomes of 12 crop plants; the As and Sb transporters were predicted to be distributed in noduline-26 like intrinsic proteins (NIPs), and every plant had one or several As and Sb transporter NIPs. Phylogenetic grouping of the NIP subfamily based on the ar/R selectivity filter and FPs were linked to As and Sb transport. We further determined the group-wise substrate selectivity profiles of the NIPs in the 12 crop plants. In addition to two NPA regions, the ar/R filter, and FPs, certain amino acids especially in the pore line, loop D, and termini contribute to the functional distinctiveness of the NIP groups. Expression analysis of transcripts in different organs indicated that most of the As and Sb transporter NIPs were expressed in roots.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.10.153DOI Listing
February 2018

Wax Ester Synthase/Diacylglycerol Acyltransferase Isoenzymes Play a Pivotal Role in Wax Ester Biosynthesis in Euglena gracilis.

Sci Rep 2017 10 18;7(1):13504. Epub 2017 Oct 18.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.
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http://dx.doi.org/10.1038/s41598-017-14077-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647427PMC
October 2017

Genome-Wide Characterization of Major Intrinsic Proteins in Four Grass Plants and Their Non-Aqua Transport Selectivity Profiles with Comparative Perspective.

PLoS One 2016 21;11(6):e0157735. Epub 2016 Jun 21.

Institute of Plant Science and Resources, Okayama University, Chuo-2-chome, Kurashiki 710-0046, Japan.

Major intrinsic proteins (MIPs), commonly known as aquaporins, transport not only water in plants but also other substrates of physiological significance and heavy metals. In most of the higher plants, MIPs are divided into five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs). Herein, we identified 68, 42, 38 and 28 full-length MIPs, respectively in the genomes of four monocot grass plants, specifically Panicum virgatum, Setaria italica, Sorghum bicolor and Brachypodium distachyon. Phylogenetic analysis showed that the grass plants had only four MIP subfamilies including PIPs, TIPs, NIPs and SIPs without XIPs. Based on structural analysis of the homology models and comparing the primary selectivity-related motifs [two NPA regions, aromatic/arginine (ar/R) selectivity filter and Froger's positions (FPs)] of all plant MIPs that have been experimentally proven to transport non-aqua substrates, we predicted the transport profiles of all MIPs in the four grass plants and also in eight other plants. Groups of MIP subfamilies based on ar/R selectivity filter and FPs were linked to the non-aqua transport profiles. We further deciphered the substrate selectivity profiles of the MIPs in the four grass plants and compared them with their counterparts in rice, maize, soybean, poplar, cotton, Arabidopsis thaliana, Physcomitrella patens and Selaginella moellendorffii. In addition to two NPA regions, ar/R filter and FPs, certain residues, especially in loops B and C, contribute to the functional distinctiveness of MIP groups. Expression analysis of transcripts in different organs indicated that non-aqua transport was related to expression of MIPs since most of the unexpressed MIPs were not predicted to facilitate the transport of non-aqua molecules. Among all MIPs in every plant, TIP (BdTIP1;1, SiTIP1;2, SbTIP2;1 and PvTIP1;2) had the overall highest mean expression. Our study generates significant information for understanding the diversity, evolution, non-aqua transport profiles and insight into comparative transport selectivity of plant MIPs, and provides tools for the development of transgenic plants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157735PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915720PMC
July 2017

Diversity and Evolution of Ascorbate Peroxidase Functions in Chloroplasts: More Than Just a Classical Antioxidant Enzyme?

Plant Cell Physiol 2016 Jul 6;57(7):1377-1386. Epub 2016 Jan 6.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504 Japan

Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of ascorbate peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of HO Current genome and phylogenetic analyses suggest that the first monofunctional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to HO A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a 'stress-sensitive phenotype' among loss-of-function mutants, at least in laboratory conditions. Interestingly, the susceptibility of enzymes to HO may have been acquired during plant evolution, thereby allowing for the flexible use of HO as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic HO as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.
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http://dx.doi.org/10.1093/pcp/pcv203DOI Listing
July 2016

Anabaena sp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers.

Proteins 2016 Jan 16;84(1):31-42. Epub 2015 Nov 16.

Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-Ku, Tokyo, 112-8681, Japan.

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.
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http://dx.doi.org/10.1002/prot.24952DOI Listing
January 2016

Biochemical and physiological analyses of NADPH-dependent thioredoxin reductase isozymes in Euglena gracilis.

Plant Sci 2015 Jul 30;236:29-36. Epub 2015 Mar 30.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo 102-0076, Japan. Electronic address:

At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol.
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http://dx.doi.org/10.1016/j.plantsci.2015.03.016DOI Listing
July 2015

Identification and functional analysis of peroxiredoxin isoforms in Euglena gracilis.

Biosci Biotechnol Biochem 2014 21;78(4):593-601. Epub 2014 May 21.

a Faculty of Life and Environmental Science, Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.

Euglena gracilis lacks catalase and contains ascorbate peroxidase (APX) which is localized exclusively in the cytosol. Other enzymes that scavenge reactive oxygen species (ROS) in Euglena have not yet been identified; therefore, ROS metabolism, especially in organelles, remains unclear in Euglena. The full-length cDNAs of four Euglena peroxiredoxins (EgPrxs) were isolated in this study. EgPrx1 and -4 were predicted to be localized in the cytosol, and EgPrx2 and -3 in plastids and mitochondria, respectively. The catalytic efficiencies of recombinant EgPrxs were similar to those of plant thiol-peroxidases, but were markedly lower than those of APX from Euglena. However, transcript levels of EgPrx1, -2, and -3 were markedly higher than those of APX. The growth rate of Euglena cells, in which the expression of EgPrx1 and -4 was suppressed by gene silencing, was markedly reduced under normal conditions, indicating physiological significance of Prx proteins.
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http://dx.doi.org/10.1080/09168451.2014.890037DOI Listing
August 2015

Expression profiles of aquaporin homologues and petal movement during petal development in Tulipa gesneriana.

Physiol Plant 2013 Jul 20;148(3):397-407. Epub 2012 Nov 20.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan.

Previously, we have characterized two tonoplast intrinsic proteins (TIPs) and four plasma membrane intrinsic proteins (PIPs) from the 2-day-old petals of tulip (Tulipa gesneriana). In this study, we analyzed the development of tulip petals and stems, temperature-dependent petal movement, the amount of ³H₂O transported into petals and stems during petal movement, and the transcript levels of two TIP (TgTIP1;1 and TgTIP1;2) and four TgPIP genes in petals and stems, from the first day of petal opening to day 12. The development of the petals and stems was completed by days 6 and 9, respectively, after the first day of petal opening. Temperature-dependent petal movement and the amount of ³H₂O that was transported into petals could be detected at significant levels up to day 6 with petal movement reaching a peak at day 3. Real-time reverse transcription-polymerase chain reaction analysis revealed that TgTIP1;1 and TgTIP1;2 were expressed ubiquitously in petals, stems, leaves, bulbs and roots. However, the expression level of TgTIP1;2 was very low in bulbs. The expression of both TgTIP1 genes was upregulated in close association with the development of petals but not with that of the stem. The four TgPIP genes were expressed at almost the same level during the development of the petals and the stem. However, the levels of the TgTIP1 and TgPIP transcripts in petals decreased during the course of petal wilting from day 9 onwards. These results suggest that TgTIP1;1 and TgTIP1;2 may contribute to petal development.
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http://dx.doi.org/10.1111/j.1399-3054.2012.01717.xDOI Listing
July 2013

Amino acid residues of both the extracellular and transmembrane domains influence binding of the antiparasitic agent milbemycin to Haemonchus contortus AVR-14B glutamate-gated chloride channels.

Biochem Biophys Res Commun 2012 Mar 17;419(3):562-6. Epub 2012 Feb 17.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan.

Glutamate-gated chloride (GluCl) channels are pentameric receptors for the inhibitory neurotransmitter glutamate in invertebrates and are a major target for macrolide anthelmintics. Three amino acids in GluCl channels are reported to render macrolide resistance in nematodes and insects. To examine whether these three amino acids are involved in binding of the antiparasitic agent milbemycin (MLM) to the GluCl channels of the nematode parasite Haemonchus contortus, the equivalent amino acids (L256, P316, and G329) of the Hco-AVR-14B subunit were substituted with various amino acids. cDNAs encoding the wild type and mutants of this subunit were transfected into COS-1 cells for transient expression and analysis of GluCl channels. The abilities of these mutant channels to bind [(3)H]MLM A(4) were remarkably decreased when compared with the wild-type channel. In patch clamp analysis, L256F and P316S mutant channels were 37- and 100-fold less sensitive to MLM A(4) when compared with the wild-type channel, respectively. These findings indicate that amino acid changes in the β10 strand, the M2-M3 linker, and the M3 region influence MLM A(4) binding to the channel. Homology modeling and ligand docking studies suggest the presence of two potential binding sites for MLM A(4).
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http://dx.doi.org/10.1016/j.bbrc.2012.02.062DOI Listing
March 2012

Analysis of two L-Galactono-1,4-lactone-responsive genes with complementary expression during the development of Arabidopsis thaliana.

Plant Cell Physiol 2012 Mar 9;53(3):592-601. Epub 2012 Feb 9.

Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504 Japan.

Unraveling the role of genes annotated as protein of unknown function is of importance in progression of plant science. l-Galactono-1,4-lactone (l-GalL) is the terminal precursor for ascorbic acid (AsA) biosynthesis in Arabidopsis thaliana, and a previous study showed two DUF (domains of unknown function) 642 family genes (At1g80240 and At5g25460, designated as DGR1 and DGR2, respectively) to be sensitive to it. In this work, leaves from wild-type Arabidopsis were fed with d-glucose, l-galactose, l-GalL and AsA, and the expression level of the At1g80240 and At5g25460 genes showed a specific response to l-GalL, but not to the other supplements despite the increases of the tissue AsA contents. Analysis of promoter-β-glucuronidase (GUS) transgenic plants showed the two genes to be complementarily expressed at the root apex and in the rest of the root excluding the apex, respectively, in both young and old seedlings, and to be expressed at the leaf primordia. The GUS activity under the control of the At5g25460 promoter was high in the cotyledon and leaf veins of young seedlings. These findings were consistent with the results of quantitative real-time PCR. Interestingly, the T-DNA insertion mutant of At5g25460 (SALK_125079) displayed shorter roots and smaller rosettes than Col-0; however, no phenotypic difference was observed between the T-DNA insertion mutant of At1g80240 and the wild type. This is the first report on the expression and functional analysis of these two DUF642 family genes, with the results revealing the contribution of DGR genes to the development of Arabidopsis.
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http://dx.doi.org/10.1093/pcp/pcs014DOI Listing
March 2012

L-aspartate dehydrogenase: features and applications.

Appl Microbiol Biotechnol 2012 Jan 27;93(2):503-16. Epub 2011 Nov 27.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

L-amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of L-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD(+)) or nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member L-aspartate dehydrogenase (L-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of L-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type L-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few L-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes.
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http://dx.doi.org/10.1007/s00253-011-3730-4DOI Listing
January 2012

Molecular cloning and sequence and 3D models analysis of the Sec61α subunit of protein translocation complex from Penicillium ochrochloron.

BMB Rep 2011 Nov;44(11):719-24

Department of Genetic Engineering & Biotechnology, Shahjalal University of Science and Technology, Bangladesh.

The Sec61α subunit is the core subunit of the protein conducting channel which is required for protein translocation in eukaryotes and prokaryotes. In this study, we cloned a Sec61α subunit from Penicillium ochrochloron (PoSec61α). Sequence and 3D structural model analysis showed that PoSec61α conserved the typical characteristics of eukaryotic and prokaryotic Sec61α subunit homologues. The pore ring known as the constriction point of the channel is formed by seven hydrophobic amino acids. Two methionine residues from transmembrane α-helice 7 (TM7) contribute to the pore ring formation and projected notably to the pore area and narrowed the pore compared with the superposed residues at the corresponding positions in the crystal structures or the 3D models of the Sec61α subunit homologues in archaea or other eukaryotes, respectively. Results reported herein indicate that the pore ring residues differ among Sec61α subunit homologues and two hydrophobic residues in the TM7 contribute to the pore ring formation.
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http://dx.doi.org/10.5483/BMBRep.2011.44.11.719DOI Listing
November 2011

Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway.

J Exp Bot 2012 Jan 6;63(1):229-39. Epub 2011 Oct 6.

Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-(14)C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits.
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http://dx.doi.org/10.1093/jxb/err275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245467PMC
January 2012

Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs.

Biochim Biophys Acta 2012 Jan 22;1818(1):1-11. Epub 2011 Sep 22.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.
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http://dx.doi.org/10.1016/j.bbamem.2011.09.014DOI Listing
January 2012

Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana.

Biosci Biotechnol Biochem 2011 7;75(9):1783-8. Epub 2011 Sep 7.

Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University.

Arabidopsis thaliana contains two GDP-L-galactose phosphorylase genes, VTC2 and VTC5, which are critical for ascorbate (AsA) biosynthesis. We investigated the expression levels of both VTC2 and VTC5 genes in wild-type A. thaliana and the AsA deficient mutants during early seedling growth. Ascorbate accumulated to an equal extent in all genotypes up to 5 d post-germination (DPG). The transcript level of VTC2 was dominant, and increased in parallel with AsA accumulation in the wild type. On the other hand, the expression of VTC5 compensated for the reduced VTC2 transcription levels in the AsA deficient mutant vtc2-1 in young seedlings. A luciferase activity assay indicated that the VTC5 promoter was more active in young (2 DPG) cotyledons and that the VTC2 and VTC5 promoters drove a day-to-night variation in expression. The present work provides clues to the precise roles of VTC2 and VTC5 in AsA biosynthesis in A. thaliana at the young seedling stage.
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http://dx.doi.org/10.1271/bbb.110320DOI Listing
January 2012

A non-NadB type L-aspartate dehydrogenase from Ralstonia eutropha strain JMP134: molecular characterization and physiological functions.

Biosci Biotechnol Biochem 2011 7;75(8):1524-32. Epub 2011 Aug 7.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Shimane, Japan.

We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis.
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http://dx.doi.org/10.1271/bbb.110216DOI Listing
January 2012

A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.

Appl Microbiol Biotechnol 2011 Jun 6;90(6):1953-62. Epub 2011 Apr 6.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, Japan.

L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.
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http://dx.doi.org/10.1007/s00253-011-3208-4DOI Listing
June 2011

Expression of aspartyl protease and C3HC4-type RING zinc finger genes are responsive to ascorbic acid in Arabidopsis thaliana.

J Exp Bot 2011 Jun 18;62(10):3647-57. Epub 2011 Mar 18.

Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

Ascorbate (AsA) is a redox buffer and enzyme cofactor with various proposed functions in stress responses and growth. The aim was to identify genes whose transcript levels respond to changes in leaf AsA. The AsA-deficient Arabidopsis mutant vtc2-1 was incubated with the AsA precursor L-galactono-1,4-lactone (L-GalL) to increase leaf AsA concentration. Differentially expressed genes screened by DNA microarray were further characterized for AsA responsiveness in wild-type plants. The analysis of 14 candidates by real-time PCR identified an aspartyl protease gene (ASP, At1g66180) and a C3HC4-type RING zinc finger gene (AtATL15, At1g22500) whose transcripts were rapidly responsive to increases in AsA pool size caused by L-GalL and AsA supplementation and light. Transgenic Arabidopsis plants expressing an AtATL15 promoter::luciferase reporter confirmed that the promoter is L-GalL, AsA, and light responsive. The expression patterns of ASP and AtATL15 suggest they have roles in growth regulation. The promoter of AtATL15 is responsive to AsA status and will provide a tool to investigate the functions of AsA in plants further.
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http://dx.doi.org/10.1093/jxb/err068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130181PMC
June 2011

Functional characterization and hyperosmotic regulation of aquaporin in Synechocystis sp. PCC 6803.

Plant Sci 2011 Feb 23;180(2):375-82. Epub 2010 Oct 23.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

The genome of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter, Synechocystis) contains an aqpZ gene (slr2057) which encodes an aquaporin (SsAqpZ), a membrane channel protein that might play a role in osmotic water transport and therefore the growth of Synechocystis. Structural characterization of SsAqpZ by protein sequence analysis and homology modelling revealed that it was more similar to bacterial aquaporin Z than the glycerol facilitator. To understand the functional role of SsAqpZ, the aqpZ knockout (KO) and myc-tagged aqpZ knockin (KI) Synechocystis were constructed. Water channel activity assays showed that SsAqpZ facilitated water transportation. SsAqpZ-mediated changes in cell volume were observed in wild-type (WT) and KI Synechocystis. Expression of SsAqpZ in KI Synechocystis was induced by extracellular hyperosmolarity. In the absence of hyperosmolarity, WT, KO and KI Synechocystis showed the same pattern of growth and no morphological or phenotypical perturbations. Under hyperosmotic condition, while the WT and also KI cells maintained a similar growth rate throughout the entire exponential phase, KO cells grew significantly slower. These results indicate that SsAqpZ has water channel activity and is involved in the adaptation and maintenance of growth of Synechocystis in a hyperosmotic environment.
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http://dx.doi.org/10.1016/j.plantsci.2010.10.010DOI Listing
February 2011

Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues.

Appl Microbiol Biotechnol 2010 Aug 27;87(5):1727-36. Epub 2010 Apr 27.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H(2)O(2). The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H(2)O(2) stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H(2)O(2), respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H(2)O(2) and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.
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http://dx.doi.org/10.1007/s00253-010-2603-6DOI Listing
August 2010

Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization.

Biochem J 2010 Feb 9;426(2):125-34. Epub 2010 Feb 9.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

Euglena gracilis lacks a catalase and contains a single APX (ascorbate peroxidase) and enzymes related to the redox cycle of ascorbate in the cytosol. In the present study, a full-length cDNA clone encoding the Euglena APX was isolated and found to contain an open reading frame encoding a protein of 649 amino acids with a calculated molecular mass of 70.5 kDa. Interestingly, the enzyme consisted of two entirely homologous catalytic domains, designated APX-N and APX-C, and an 102 amino acid extension in the N-terminal region, which had a typical class II signal proposed for plastid targeting in Euglena. A computer-assisted analysis indicated a novel protein structure with an intramolecular dimeric structure. The analysis of cell fractionation showed that the APX protein is distributed in the cytosol, but not the plastids, suggesting that Euglena APX becomes mature in the cytosol after processing of the precursor. The kinetics of the recombinant mature FL (full-length)-APX and the APX-N and APX-C domains with ascorbate and H2O2 were almost the same as that of the native enzyme. However, the substrate specificity of the mature FL-APX and the native enzyme was different from that of APX-N and APX-C. The mature FL-APX, but not the truncated forms, could reduce alkyl hydroperoxides, suggesting that the dimeric structure is correlated with substrate recognition. In Euglena cells transfected with double-stranded RNA, the silencing of APX expression resulted in a significant increase in the cellular level of H2O2, indicating the physiological importance of APX to the metabolism of H2O2.
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http://dx.doi.org/10.1042/BJ20091406DOI Listing
February 2010

Molecular characterization of a novel peroxidase from the cyanobacterium Anabaena sp. strain PCC 7120.

Appl Environ Microbiol 2009 Dec 2;75(23):7509-18. Epub 2009 Oct 2.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.

The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35 degrees C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 muM.min(-1), respectively. The apparent K(m) and k(cat)/K(m) values for Reactive Blue 5 were 3.6 muM and 1.2 x 10(7) M(-1) s(-1), respectively, while the apparent K(m) and k(cat)/K(m) values for H(2)O(2) were 5.8 muM and 6.6 x 10(6) M(-1) s(-1), respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to approximately 50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.
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http://dx.doi.org/10.1128/AEM.01121-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786418PMC
December 2009

Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

Appl Environ Microbiol 2009 May 27;75(9):2792-7. Epub 2009 Feb 27.

Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu, Shimane 690-8504, Japan.

Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.
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http://dx.doi.org/10.1128/AEM.02335-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681690PMC
May 2009
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