Biochemical Characterization of Larix kaempferi Glutathione Reductase

WANG Xin; QIAN Ting-ting; ZENG Qing-yin

Volume 26 Issue S1

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Biochemical Characterization of Larix kaempferi Glutathione Reductase

1.
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
2.
University of Chinese Academy of Sciences, Beijing 100049, China

Received Date: 2013-08-20

Abstract

Glutathione reductases (GRs) are key enzymes in plant glutathione-ascorbate cycle, and play important roles in plant oxidative stress tolerance. In this study, a GR gene (LaGR) was cloned from Larix kaempferi. This gene encodes protein of 563 amino acid residues, with calculated molecular masses of 61.06 kDa. RT-PCR reveals that LaGR is a constitutive expression gene, which is expressed in all tissues detected, including bud, mature needle, phloem of stem and root. The chloroplast localization of LaGR was confirmed by transient expression in Arabidopsis protoplast. LaGR was overexpressed in Escherichia coli, and purified by Ni-affinity chromatography. The purified LaGR proteins showed high catalytic activities and affinities towards substrates GSSG and NADPH. LaGR protein is a thermostable protein, and has optimal pH ranging from 7.0 to 9.0. Heavy metal ions (Cd2+, Pb2+ and Cu2+) could inhibit the LaGR's activities. When the His528 of LaGR protein was replaced by Gln, the mutant proteins showed decreased enzymatic activities and increased affinity towards substrates GSSG and NADPH. Moreover, the mutant protein showed less thermostability than wild-type protein. Thus, His528 of LaGR protein might contribute to the enzyme's catalytic activity and structural stability.
- Larix kaempferi,
- glutathione reductase,
- expression pattern,
- subcellular localization,
- enzymatic characterization

References

[1]	Rao A S V C, Reddy A R. Glutathione reductase: a putative redox regulatory system in plant cells[M]. Sulfur Assimilation and Abiotic Stress in Plants, Netherlands: Springer, 2008: 111-147
[2]	Kim I S, Kim Y S, Yoon H S. Glutathione reductase from Oryza sativa increases acquired tolerance to abiotic stresses in a genetically modified Saccharomyces cerevisiae strain[J]. Journal of Microbiology and Biotechnology, 2012, 22(11): 1557-1567
[3]	Foyer C H, Souriau N, Perret S, et al. Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees[J].Plant Physiology, 1995,109(3): 1047-1057
[4]	Chen Y P, Xing L P, Wu G J, et al. Plastidial glutathione reductase from Haynaldia villosa is an enhancer of powdery mildew resistance in wheat (Triticum aestivum)[J].Plant and Cell Physiology, 2007,48(12): 1702-1712
[5]	吴涛,张俊红,韩素英,等. 日本落叶松小 RNA 文库构建及其 microRNA 鉴定[J].林业科学研究, 2012,25(6): 677-684
[6]	Tang Z X, Yang H L. Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa[J].Molecular Biology Reports, 2013, 40: 5105-5114
[7]	Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis [J].Nature Protocols, 2007, 2(7): 1565-1572
[8]	Mavis R D, Stellwagen E. Purification and subunit structure of glutathione reductase from bakers'yeast[J].Journal of Biological Chemistry, 1968, 243(4): 809-814
[9]	Rouhier N, Couturier J, Jacquot J P. Genome-wide analysis of plant glutaredoxin systems[J].Journal of Experimental Botany, 2006,57(8): 1685-1696
[10]	Marty L, Siala W, Schwarzlander M, et al. The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis[J].Proceedings of the National Academy of Sciences, 2009, 106(22): 9109-9114
[11]	Lascano H R, Casano L M, Melchiorre M N, et al. Biochemical and molecular characterisation of wheat chloroplastic glutathione reductase[J].Biologia plantarum, 2001,44(4): 509-516
[12]	Kalt-Torres W, Burke J J, Anderson J M. Chloroplast glutathione reductase: purification and properties[J].Physiologia Plantarum, 1984, 61(2): 271-278
[13]	Connell J P, Mullet J E. Pea chloroplast glutathione reductase: purification and characterization[J].Plant Physiology, 1986,82(2): 351-356
[14]	Mahan J R, Burke J J. Purification and characterization of glutathione reductase from corn mesophyll chloroplasts [J].Physiologia Plantarum, 1987, 71(3): 352-358
[15]	LóPEZ-BAREA J, LEE C Y. Mouse-liver glutathione reductase [J].European Journal of Biochemistry, 1979, 98(2): 487-499
[16]	Rescigno M, Perham R N. Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution [J].Biochemistry, 1994, 33(19): 5721-5727
[17]	Coban T A, Senturk M, Ciftci M, et al. Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study[J].Protein and Peptide Letters, 2007, 14(10): 1027-1030
[18]	Tekman B, Ozdemir H, Senturk M, et al. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity [J].Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2008, 148(2): 117-121
[19]	Deonarain M P, Berry A, Scrutton N S, et al. Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99 [J]. Biochemistry, 1989, 28(25): 9602-9607

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Biochemical Characterization of Larix kaempferi Glutathione Reductase

1. State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

2. University of Chinese Academy of Sciences, Beijing 100049, China

Received Date: 2013-08-20

Abstract: Glutathione reductases (GRs) are key enzymes in plant glutathione-ascorbate cycle, and play important roles in plant oxidative stress tolerance. In this study, a GR gene (LaGR) was cloned from Larix kaempferi. This gene encodes protein of 563 amino acid residues, with calculated molecular masses of 61.06 kDa. RT-PCR reveals that LaGR is a constitutive expression gene, which is expressed in all tissues detected, including bud, mature needle, phloem of stem and root. The chloroplast localization of LaGR was confirmed by transient expression in Arabidopsis protoplast. LaGR was overexpressed in Escherichia coli, and purified by Ni-affinity chromatography. The purified LaGR proteins showed high catalytic activities and affinities towards substrates GSSG and NADPH. LaGR protein is a thermostable protein, and has optimal pH ranging from 7.0 to 9.0. Heavy metal ions (Cd2+, Pb2+ and Cu2+) could inhibit the LaGR's activities. When the His528 of LaGR protein was replaced by Gln, the mutant proteins showed decreased enzymatic activities and increased affinity towards substrates GSSG and NADPH. Moreover, the mutant protein showed less thermostability than wild-type protein. Thus, His528 of LaGR protein might contribute to the enzyme's catalytic activity and structural stability.

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[1]	Rao A S V C, Reddy A R. Glutathione reductase: a putative redox regulatory system in plant cells[M]. Sulfur Assimilation and Abiotic Stress in Plants, Netherlands: Springer, 2008: 111-147
[2]	Kim I S, Kim Y S, Yoon H S. Glutathione reductase from Oryza sativa increases acquired tolerance to abiotic stresses in a genetically modified Saccharomyces cerevisiae strain[J]. Journal of Microbiology and Biotechnology, 2012, 22(11): 1557-1567
[3]	Foyer C H, Souriau N, Perret S, et al. Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees[J].Plant Physiology, 1995,109(3): 1047-1057
[4]	Chen Y P, Xing L P, Wu G J, et al. Plastidial glutathione reductase from Haynaldia villosa is an enhancer of powdery mildew resistance in wheat (Triticum aestivum)[J].Plant and Cell Physiology, 2007,48(12): 1702-1712
[5]	吴涛,张俊红,韩素英,等. 日本落叶松小 RNA 文库构建及其 microRNA 鉴定[J].林业科学研究, 2012,25(6): 677-684
[6]	Tang Z X, Yang H L. Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa[J].Molecular Biology Reports, 2013, 40: 5105-5114
[7]	Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis [J].Nature Protocols, 2007, 2(7): 1565-1572
[8]	Mavis R D, Stellwagen E. Purification and subunit structure of glutathione reductase from bakers'yeast[J].Journal of Biological Chemistry, 1968, 243(4): 809-814
[9]	Rouhier N, Couturier J, Jacquot J P. Genome-wide analysis of plant glutaredoxin systems[J].Journal of Experimental Botany, 2006,57(8): 1685-1696
[10]	Marty L, Siala W, Schwarzlander M, et al. The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis[J].Proceedings of the National Academy of Sciences, 2009, 106(22): 9109-9114
[11]	Lascano H R, Casano L M, Melchiorre M N, et al. Biochemical and molecular characterisation of wheat chloroplastic glutathione reductase[J].Biologia plantarum, 2001,44(4): 509-516
[12]	Kalt-Torres W, Burke J J, Anderson J M. Chloroplast glutathione reductase: purification and properties[J].Physiologia Plantarum, 1984, 61(2): 271-278
[13]	Connell J P, Mullet J E. Pea chloroplast glutathione reductase: purification and characterization[J].Plant Physiology, 1986,82(2): 351-356
[14]	Mahan J R, Burke J J. Purification and characterization of glutathione reductase from corn mesophyll chloroplasts [J].Physiologia Plantarum, 1987, 71(3): 352-358
[15]	LóPEZ-BAREA J, LEE C Y. Mouse-liver glutathione reductase [J].European Journal of Biochemistry, 1979, 98(2): 487-499
[16]	Rescigno M, Perham R N. Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution [J].Biochemistry, 1994, 33(19): 5721-5727
[17]	Coban T A, Senturk M, Ciftci M, et al. Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study[J].Protein and Peptide Letters, 2007, 14(10): 1027-1030
[18]	Tekman B, Ozdemir H, Senturk M, et al. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity [J].Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2008, 148(2): 117-121
[19]	Deonarain M P, Berry A, Scrutton N S, et al. Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99 [J]. Biochemistry, 1989, 28(25): 9602-9607

Biochemical Characterization of Larix kaempferi Glutathione Reductase

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通讯作者: 陈斌, bchen63@163.com

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Biochemical Characterization of Larix kaempferi Glutathione Reductase

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