Prokaryotic Expression, Purification and Enzyme Activity Assay of Thymidylate Kinase of the Paulownia Witches'-broom Phytoplasma

SONG Chuan-sheng; HU Jia-xu; LIN Cai-li; REN Zheng-guang; GENG Xian-sheng; TIAN Guo-zhong

Volume 27 Issue 6

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Prokaryotic Expression, Purification and Enzyme Activity Assay of Thymidylate Kinase of the Paulownia Witches'-broom Phytoplasma

1.
Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Key Laboratory of Forest Protection, State Forestry Administration, Beijing 100091, China
2.
Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin 300457, China
3.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China

Received Date: 2014-03-11

Abstract

Thymidylate kinase catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis, and it is crucial for DNA replication and life survival. In previous study, the tmk-a-1, tmk-a-2 and tmk-b genes of Paulownia witches'-broom Pingshan strain (PaWBPS) were obtained. On the basis of previous study, the amino acid sequence alignment and similarity analysis were conducted among PaWBPS TMK-a-1, PaWBPS TMK-a-2, PaWBPS TMK-b, onion yellow wild-type line (OY-W) TMK-a, OY-W TMK-b, white blue dwarf (WBD) TMK-a and WBD TMK-b. The amino acid sequence similarity value was 96.65% between PaWBPS TMK-b and WBD TMK-2, 99.03% between PaWBPS TMK-b and OY-W TMK-b, 90.57% between PaWBPS TMK-a-1 and PaWBPS TMK-a-2, ranged from 87.32% to 99.26% among PaWBPS TMK-a-1, PaWBPS TMK-a-2, WBD TMK-1 and OY-W TMK-a, and from 22.22% to 25.95% among TMK-a and TMK-b of PaWBPS, OY-W and WBD phytoplasma. The pET28a system was used to generate a polyhistidine (polyHis)-tagged TMK fusion protein. The polyHis-tagged TMKs were expressed in Escherichia coli BL21 (DE3) cell and purified under native conditions by Ni-NTA chelating column. Then, the TMK activity was measured using enzyme-coupled assay. Results suggested that the TMK-b had thymidylate kinase activity (85.96±0.74 U·mg-1) and the TMK-a-1 and TMK-a-2 had hardly any activity.
- Paulownia witches'-broom phytoplasma,
- thymidylate kinase,
- prokaryotic expression,
- enzyme activity

References

[1]	Tian G Z, Raychauduri, S P. Paulownia witches' broom disease in China: present status. In Forest Trees and Palms:Diseases and Control [M], Oxford, IBH, 1996: 227-251.
[2]	Gesing A, Modrzejewska H, Kathowmik M, et al. Thymidine kinase and adenosine kinase activities in homogenates of thyroid lobes in hemithy-roidectomized rates affects of melatonim in vitro [J]. neuroendocrinology letters, 2000, 21(6):453-459.
[3]	Pallack J, Williams M, Mcelhaney R, et al. The comparative metabolism of the mollicutes: the utility for taxonomic classification and the relationship of pupative gene annotation and hylogeny to enzymatic function in the smallest frii-living cells [J]. Critical Reviews in Microbiology, 1997, 23:269-354.
[4]	Ronceret A, Gadea-Vacas J, Guilleminot J, et al. The first zygotic division in Arabidopsis requires de novo transcription of thymidylate kinase [J]. The Plant Journal, 2008, 53: 776-789.
[5]	Sclafani R A, Fangman W L. Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK [J]. Proceedings of the National Academy of the Sciences of the United States of America, 1984, 81: 5821-5825.
[6]	White J H, Green S R, Barker D G, et al. The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription [J]. Experimental Cell Research, 1987, 171: 223-231.
[7]	Bueno R V, Braya R C, Segretti N D, et al. New Tuberculostatic agents targeting nucleic acid biosynthesis: drug design using QSAR approaches [J]. Current Pharmaceutical Design, 2014, 20: 4474- 4485
[8]	Doharey P K, Suthar M K, Verma A, et al. Molecular cloning and characterization of Bruyia malayi thymidylate kinase [J]. Acta Tropica, 2014, 133:83-92.
[9]	Guinaraes A P, Ramolho T C, Franca T E. Preventing the return of smallpox: molecular modeling studies on thymidylate kinase from Variola virus [J]. Journal of Biomolecular structure & Dynamics, 2013, DOI: 10.1080/07391102.2013.830578.
[10]	江施幸. 丝瓜簇叶病植物菌质体胸苷酸激酶基因 (tmk) 之表现及特性研究[D]. 国立中央大学硕士论文, 2001.
[11]	Miyata S, Oshima K, Kakizawa S, et al. Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded iin the onion yellows phytoplasma genome [J]. Microbiology, 2003, 149: 2243-2250.
[12]	李蓓, 纪玲玲, 吴云峰, 等. 小麦蓝矮病植原体胸苷酸激酶基因的分离、原核表达及酶活性分析 [J]. 微生物学报, 2008, 48(6):739-744.
[13]	Bai X, Zhang J, Ewing A. Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts [J]. Journal of Bacteriology, 2006, 188 (10): 3682-3696.
[14]	Jomantiene R, Davis R E. Clusters of diverse genes existing as multiple, sequence-variable mosaics in a phytoplasma genome [J]. Fems Microbiology Letters, 2006, 255 (1):59-65.
[15]	宋传生, 胡佳续, 林彩丽, 等. 泡桐丛枝植原体胸苷酸激酶基因的克隆及序列分析[C]. 中国植物病理学会2012年学术年会论文集, 2013:303.
[16]	田国忠, 温秀军, 李永, 等. 枣疯病和泡桐丛枝病植原体不同分离物组织培养保藏和嫁接传病试验比较 [J]. 林业科学研究, 2005, 18(1):1-9.
[17]	Berghauser J. A reactive arginine in adenylate kinase [J]. Biochimica et Biophysica Acta (BBA) - Enzymology, 1975, 397:370-376.
[18]	李淑娟. 六聚组氨酸融合蛋白纯化方法研究. 西北大学硕士学位论文, 2007.
[19]	Wang L, Westberg J, Bolske G, et al. Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: ecidence for efficient utilization of deoxynucleosides [J]. Molecular Microbiology, 2001, 42:1065-1073.
[20]	Collins CM, Olstad K, Sterud E, et al. Isolation of a novel fish thymidylate kinase gene, upregulated in Atlantic salmon (Salmo salar L.) following infection with the monogenean parasite Gyrodactylus salaries [J]. Fish and Shellfish Immunology, 2007, 23:793-807.
[21]	Muhier-Lehmann H, Chaffotte A, Pochet S, et al. Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes [J]. Protein Science, 2001, 10:1195-1205.
[22]	Brundiers R, Lavie A, Veit T, et al. Modifying human thymidylate kinase to potentiate azidothymidine activation [J]. Journal of Biological Chemistry, 1999, 274:35289-35292.
[23]	Reynes J P, Tiraby M, Baron M, et al. Escherichia coli thymidylate Kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk Locus [J]. Journal of Bacteriology, 1996, 178:2804-2812.
[24]	Balasubramaniam N K, Veerisetty V, Gentry G A. Herpesviral deoxythymidine kinases contain a site analogous to the phosphoryl-binding arginine-rich region of porcine adenylate kinase; comparison of secondary structure predictions and conservation [J]. Journal of General Virology, 1990, 71:2979-2987.
[25]	Chaperon D N. Construction and complementation of in-frame deletions of the essential Escherichia coli thymidylate kinase gene [J]. Applied and Environmental Microbiology, 2006, 72:1288-1294.
[26]	徐启聪. 中国不同地区不同枣树品种上枣疯病植原体的分子鉴定及变异分析[D]. 中国林业科学研究院硕士学位论文, 2009.
[27]	Ostermann N, Schlichting I, Brundier R, et al. Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexws along the reaction coordinate [J]. Structure, 2000, 8:629-642.
[28]	Tourneux L, Bucurenci N, Lascu I, et al. Substituttion of an alanine residue for glycine 146 in TMP kinase from Escherichia coli responsible for bacterial hypersensitivity to bromodeoxyuridine [J]. Journal of Bacteriology, 1998, 180(16):4291-4293.
[29]	牟海青, 周涛, 赵文军, 等. 泡桐丛枝植原体抗原膜蛋白抗血清的制备及应用 [J]. 植物病理学报, 2011, 41(2):161-170.
[30]	Wang J, Zhu X P, Gao R, et al. Genetic and serological analyses of elongation factor EF-Tu of paulownia witches'-broom phytoplasma (16SrI-D) [J]. Plant Pathology, 2010, 59:972-981.

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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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Prokaryotic Expression, Purification and Enzyme Activity Assay of Thymidylate Kinase of the Paulownia Witches'-broom Phytoplasma

1. Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Key Laboratory of Forest Protection, State Forestry Administration, Beijing 100091, China

2. Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin 300457, China

3. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China

Received Date: 2014-03-11

Abstract: Thymidylate kinase catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis, and it is crucial for DNA replication and life survival. In previous study, the tmk-a-1, tmk-a-2 and tmk-b genes of Paulownia witches'-broom Pingshan strain (PaWBPS) were obtained. On the basis of previous study, the amino acid sequence alignment and similarity analysis were conducted among PaWBPS TMK-a-1, PaWBPS TMK-a-2, PaWBPS TMK-b, onion yellow wild-type line (OY-W) TMK-a, OY-W TMK-b, white blue dwarf (WBD) TMK-a and WBD TMK-b. The amino acid sequence similarity value was 96.65% between PaWBPS TMK-b and WBD TMK-2, 99.03% between PaWBPS TMK-b and OY-W TMK-b, 90.57% between PaWBPS TMK-a-1 and PaWBPS TMK-a-2, ranged from 87.32% to 99.26% among PaWBPS TMK-a-1, PaWBPS TMK-a-2, WBD TMK-1 and OY-W TMK-a, and from 22.22% to 25.95% among TMK-a and TMK-b of PaWBPS, OY-W and WBD phytoplasma. The pET28a system was used to generate a polyhistidine (polyHis)-tagged TMK fusion protein. The polyHis-tagged TMKs were expressed in Escherichia coli BL21 (DE3) cell and purified under native conditions by Ni-NTA chelating column. Then, the TMK activity was measured using enzyme-coupled assay. Results suggested that the TMK-b had thymidylate kinase activity (85.96±0.74 U·mg-1) and the TMK-a-1 and TMK-a-2 had hardly any activity.

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Reference (30)

[1]	Tian G Z, Raychauduri, S P. Paulownia witches' broom disease in China: present status. In Forest Trees and Palms:Diseases and Control [M], Oxford, IBH, 1996: 227-251.
[2]	Gesing A, Modrzejewska H, Kathowmik M, et al. Thymidine kinase and adenosine kinase activities in homogenates of thyroid lobes in hemithy-roidectomized rates affects of melatonim in vitro [J]. neuroendocrinology letters, 2000, 21(6):453-459.
[3]	Pallack J, Williams M, Mcelhaney R, et al. The comparative metabolism of the mollicutes: the utility for taxonomic classification and the relationship of pupative gene annotation and hylogeny to enzymatic function in the smallest frii-living cells [J]. Critical Reviews in Microbiology, 1997, 23:269-354.
[4]	Ronceret A, Gadea-Vacas J, Guilleminot J, et al. The first zygotic division in Arabidopsis requires de novo transcription of thymidylate kinase [J]. The Plant Journal, 2008, 53: 776-789.
[5]	Sclafani R A, Fangman W L. Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK [J]. Proceedings of the National Academy of the Sciences of the United States of America, 1984, 81: 5821-5825.
[6]	White J H, Green S R, Barker D G, et al. The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription [J]. Experimental Cell Research, 1987, 171: 223-231.
[7]	Bueno R V, Braya R C, Segretti N D, et al. New Tuberculostatic agents targeting nucleic acid biosynthesis: drug design using QSAR approaches [J]. Current Pharmaceutical Design, 2014, 20: 4474- 4485
[8]	Doharey P K, Suthar M K, Verma A, et al. Molecular cloning and characterization of Bruyia malayi thymidylate kinase [J]. Acta Tropica, 2014, 133:83-92.
[9]	Guinaraes A P, Ramolho T C, Franca T E. Preventing the return of smallpox: molecular modeling studies on thymidylate kinase from Variola virus [J]. Journal of Biomolecular structure & Dynamics, 2013, DOI: 10.1080/07391102.2013.830578.
[10]	江施幸. 丝瓜簇叶病植物菌质体胸苷酸激酶基因 (tmk) 之表现及特性研究[D]. 国立中央大学硕士论文, 2001.
[11]	Miyata S, Oshima K, Kakizawa S, et al. Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded iin the onion yellows phytoplasma genome [J]. Microbiology, 2003, 149: 2243-2250.
[12]	李蓓, 纪玲玲, 吴云峰, 等. 小麦蓝矮病植原体胸苷酸激酶基因的分离、原核表达及酶活性分析 [J]. 微生物学报, 2008, 48(6):739-744.
[13]	Bai X, Zhang J, Ewing A. Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts [J]. Journal of Bacteriology, 2006, 188 (10): 3682-3696.
[14]	Jomantiene R, Davis R E. Clusters of diverse genes existing as multiple, sequence-variable mosaics in a phytoplasma genome [J]. Fems Microbiology Letters, 2006, 255 (1):59-65.
[15]	宋传生, 胡佳续, 林彩丽, 等. 泡桐丛枝植原体胸苷酸激酶基因的克隆及序列分析[C]. 中国植物病理学会2012年学术年会论文集, 2013:303.
[16]	田国忠, 温秀军, 李永, 等. 枣疯病和泡桐丛枝病植原体不同分离物组织培养保藏和嫁接传病试验比较 [J]. 林业科学研究, 2005, 18(1):1-9.
[17]	Berghauser J. A reactive arginine in adenylate kinase [J]. Biochimica et Biophysica Acta (BBA) - Enzymology, 1975, 397:370-376.
[18]	李淑娟. 六聚组氨酸融合蛋白纯化方法研究. 西北大学硕士学位论文, 2007.
[19]	Wang L, Westberg J, Bolske G, et al. Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: ecidence for efficient utilization of deoxynucleosides [J]. Molecular Microbiology, 2001, 42:1065-1073.
[20]	Collins CM, Olstad K, Sterud E, et al. Isolation of a novel fish thymidylate kinase gene, upregulated in Atlantic salmon (Salmo salar L.) following infection with the monogenean parasite Gyrodactylus salaries [J]. Fish and Shellfish Immunology, 2007, 23:793-807.
[21]	Muhier-Lehmann H, Chaffotte A, Pochet S, et al. Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes [J]. Protein Science, 2001, 10:1195-1205.
[22]	Brundiers R, Lavie A, Veit T, et al. Modifying human thymidylate kinase to potentiate azidothymidine activation [J]. Journal of Biological Chemistry, 1999, 274:35289-35292.
[23]	Reynes J P, Tiraby M, Baron M, et al. Escherichia coli thymidylate Kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk Locus [J]. Journal of Bacteriology, 1996, 178:2804-2812.
[24]	Balasubramaniam N K, Veerisetty V, Gentry G A. Herpesviral deoxythymidine kinases contain a site analogous to the phosphoryl-binding arginine-rich region of porcine adenylate kinase; comparison of secondary structure predictions and conservation [J]. Journal of General Virology, 1990, 71:2979-2987.
[25]	Chaperon D N. Construction and complementation of in-frame deletions of the essential Escherichia coli thymidylate kinase gene [J]. Applied and Environmental Microbiology, 2006, 72:1288-1294.
[26]	徐启聪. 中国不同地区不同枣树品种上枣疯病植原体的分子鉴定及变异分析[D]. 中国林业科学研究院硕士学位论文, 2009.
[27]	Ostermann N, Schlichting I, Brundier R, et al. Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexws along the reaction coordinate [J]. Structure, 2000, 8:629-642.
[28]	Tourneux L, Bucurenci N, Lascu I, et al. Substituttion of an alanine residue for glycine 146 in TMP kinase from Escherichia coli responsible for bacterial hypersensitivity to bromodeoxyuridine [J]. Journal of Bacteriology, 1998, 180(16):4291-4293.
[29]	牟海青, 周涛, 赵文军, 等. 泡桐丛枝植原体抗原膜蛋白抗血清的制备及应用 [J]. 植物病理学报, 2011, 41(2):161-170.
[30]	Wang J, Zhu X P, Gao R, et al. Genetic and serological analyses of elongation factor EF-Tu of paulownia witches'-broom phytoplasma (16SrI-D) [J]. Plant Pathology, 2010, 59:972-981.

Prokaryotic Expression, Purification and Enzyme Activity Assay of Thymidylate Kinase of the Paulownia Witches'-broom Phytoplasma

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通讯作者: 陈斌, bchen63@163.com

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Prokaryotic Expression, Purification and Enzyme Activity Assay of Thymidylate Kinase of the Paulownia Witches'-broom Phytoplasma

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