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[1]刘辉,刘璋敏,冯雁,等.链霉菌来源谷氨酰胺合成酶GLnA_Shy的性质表征[J].南京工业大学学报(自然科学版),2020,42(02):249-256.[doi:10.3969/j.issn.1671-7627.2020.02.017]
 LIU Hui,LIU Zhangmin,FENG Yan,et al.Characterization of glutamine synthetase GLnA_Shy from Streptomyces[J].Journal of NANJING TECH UNIVERSITY(NATURAL SCIENCE EDITION),2020,42(02):249-256.[doi:10.3969/j.issn.1671-7627.2020.02.017]
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链霉菌来源谷氨酰胺合成酶GLnA_Shy的性质表征()
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《南京工业大学学报(自然科学版)》[ISSN:1671-7627/CN:32-1670/N]

卷:
42
期数:
2020年02期
页码:
249-256
栏目:
出版日期:
2020-03-22

文章信息/Info

Title:
Characterization of glutamine synthetase GLnA_Shy from Streptomyces
文章编号:
1671-7627(2020)02-0249-08
作者:
刘辉1刘璋敏1冯雁1李谦2崔莉1
1.上海交通大学 生命科学技术学院 微生物代谢国家重点实验室,上海 200240; 2.中国药科大学 生命科学与技术学院,江苏 南京 210009
Author(s):
LIU Hui1LIU Zhangmin1FENG Yan1LI Qian2CUI Li1
1.State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China; 2. School of Life Sciences and Technology, China Pharmaceutical University, Nanjing 210009, China
关键词:
谷氨酰胺合成酶 大肠杆菌 同源建模 链霉菌 克隆表达
Keywords:
glutamine synthase Escherichia coli homology modeling Streptomyces expression
分类号:
Q814
DOI:
10.3969/j.issn.1671-7627.2020.02.017
文献标志码:
A
摘要:
在大肠杆菌BL21(DE3)中对吸水链霉菌5008的GLny_Shy基因进行克隆表达。GLnA_Shy蛋白由469个氨基酸组成,其分子量约为5.2×104。体外酶学性质及酶动力学测定结果显示,GLnA_Shy酶催化反应的最适温度为35 ℃,最适pH为7.5,对底物谷氨酸的米氏常数(Km)和催化常数(kcat)分别为(21.8±3.2)μmol/L和(3.8±0.5)×10-3 s-1。进化树分析和同源建模结果显示GLnA_Shy蛋白属于谷氨酰胺合成酶(GS)家族,该酶包含一个较小的N端结构域(残基区域为16~94)和一个较大的C端结构域(残基区域为101~466),其三级结构具有GS典型的双通道结构,与底物谷氨酸的结合位点为Glu131、Ser264、Arg320和Arg359。谷氨酰胺合成酶在含氮化合物的生产上应用广泛,通过解析GLnA_Shy的功能,为进一步对其进行定点改造提供了理论基础。
Abstract:
GLnA_Shy protein contains 469 amino acids with a molecular weight of 5.2×104. The gene GLnA_Shy from Streptomyces hygroscopicus 5008 was expressed in Escherichia coli BL21(DE3)which encoded protein GLnA_Shy. Results of enzymatic kinetics showed that the optimum temperature and pH of GLnA_Shy protein catalyzing the conversion of glutamate to glutamine were 35 ℃ and 7.5, respectively. The michaelis constant(Km)and catalytic rate constant(kcat)of GLnA_Shy were(21.8±3.2)μmol/L and(3.8±0.5)×10-3 s-1, respectively. The results of phylogenetic analysis and homology modeling indicated that GLnA_Shy protein belonged to glutamine synthase(GS)family. It had a smaller N-terminal domain(amino acid residues from 16 to 94)and a larger C-terminal domain(amino acid residues from 101 to 466), and had a classical bifunnel structure of GS. The predicted key amino acids of its binding sites with glutamate were Glu131, Ser264, Arg320 and Arg359. The functional analyses of GLnA_Shy offered a foundation for further site-specific protein modification.

参考文献/References:

[1] MAGASANIK B.The regulation of nitrogen utilization in enteric bacteria[J].Journal of Cellular Biochemistry,1993,51(1):34.
[2] PESOLE G,BOZZETTI M P,LANAVE C,et al.Glutamine synthetase gene evolution:a good molecular clock[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(2):522.
[3] KUMADA Y,BENSON D R,HILLEMANN D,et al.Evolution of the glutamine synthetase gene,one of the oldest existing and functioning genes[J].Proceedings of the National Academy of Sciences of the United States of America,1993,90(7):3009.
[4] EISENBERG D,GILL H S,PFLUEGL G M,et al.Structure-function relationships of glutamine synthetases[J].Biochimica et Biophysica Acta,2000,1477(1/2):122.
[5] ALMASSY R J,JANSON C A,HAMLIN R,et al.Novel subunit-subunit interactions in the structure of glutamine synthetase[J].Nature,1986,323:304.
[6] BROWN J R,MASUCHI Y,ROBB F T,et al.Evolutionary relationships of bacterial and archaeal glutamine synthetase genes[J].Journal of Molecular Evolution,1994,38(6):566.
[7] MATHIS R,GAMAS P,MEYER Y,et al.The presence of GSI-like genes in higher plants:support for the paralogous evolution of GSI and GSII genes[J].Journal of Molecular Evolution,2000,50(2):116.
[8] WYATT K,WHITE H E,WANG L,et al.Lengsin is a survivor of an ancient family of class I glutamine synthetases re-engineered by evolution for a role in the vertebrate lens[J].Structure,2006,14(12):1823.
[9] KRAJEWSKI W W,COLLINS R,HOLMBERG-SCHIAVONE L,et al.Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design[J].Journal of Molecular Biology,2008,375(1):217.
[10] UNNO H,UCHIDA T,SUGAWARA H,et al.Atomic structure of plant glutamine synthetase:a key enzyme for plant productivity[J].Journal of Biological Chemistry,2006,281(39):29287.
[11] VAN ROOYEN J M,ABRATT V R,BELRHALI H,et al.Crystal structure of Type Ⅲ glutamine synthetase:surprising reversal of the inter-ring interface[J].Structure,2011,19(4):471.
[12] CHEN Q Y,CHEN G A,XUE B,et al.High efficiency of L-glutamine production by coupling genetic engineered bacterial glutamine synthetase with yeast alcoholic fermentation system[J].Chinese Journal of Biotechnology,2004,20(3):456.
[13] TARDITO S,OUDIN A,AHMED S U,et al.Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma[J].Nature Cell Biology,2015,17(12):1556.
[14] SEABRA A R,PEREIRA P A,BECKER J D,et al.Inhibition of glutamine synthetase by phosphinothricin leads to transcriptome reprograming in root nodules of Medicago truncatula[J].Molecular Plant(Microbe Interactions),2012,25(7):976.
[15] MU W,ZHANG T,JIANG B.An overview of biological production of L-theanine[J].Biotechnology Advances,2015,33(3/4):335.
[16] LILLO C.Diurnal variations of nitrite reductase,glutamine synthetase,glutamate synthase,alanine aminotransferase and aspartate aminotransferase in barley leaves[J].Physiologia Plantarum,1984,61(2):214.
[17] ROBERT X,GOUET P.Deciphering key features in protein structures with the new ENDscript server[J].Nucleic Acids Research,2014,42:320.
[18] ROY A,KUCUKURAL A,ZHANG Y.I-TASSER:a unified platform for automated protein structure and function prediction[J].Nature Protocols,2010,5(4):725.
[19] YANG J Y,ZHANG Y.I-TASSER server:new development for protein structure and function predictions[J].Nucleic Acids Research,2015,43:174.
[20] ZHANG C X,FREDDOLINO P L,ZHANG Y.COFACTOR:improved protein function prediction by combining structure,sequence and protein-protein interaction information[J].Nucleic Acids Research,2017,45:291.
[21] FISHER S H,WRAY L V.Novel trans-acting bacillus subtilis glnA mutations that derepress glnRA expression[J].Journal of Bacteriology,2009,191(8):2485.
[22] RHEE S G,CHOCK P B,WEDLER F C,et al.Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. evidence from methionine sulfoximine inhibition studies[J].Journal of Biological Chemistry,1981,256(2):644.
[23] WRAY L V,FISHER S H.Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase[J].Molecular Microbiology,2008,68(2):277.
[24] MURRAY D S,CHINNAM N,TONTHAT N K,et al.Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism[J].Journal of Biological Chemistry,2013,288(50):35801.
[25] WRAY L V,FISHER S H.Functional roles of the conserved Glu304 loop of Bacillus subtilis glutamine synthetase[J].Journal of Bacteriology,2010,192(19):5018.
[26] MEHTA R,PEARSON J T,MAHAJAN S,et al.Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria[J].The Journal of Biological Chemistry,2004,279(21):22477.

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备注/Memo

备注/Memo:
收稿日期:2019-04-23
基金项目:国家自然科学基金(31770098,31620103901)
作者简介:刘辉(1992—),男,E-mail:15705276231@163.com; 崔莉(联系人),副研究员,E-mail:cuili@sjtu.edu.cn.
引用格式:刘辉,刘璋敏,冯雁,等.链霉菌来源谷氨酰胺合成酶GLnA_Shy的性质表征[J].南京工业大学学报(自然科学版),2020,42(2):249-256.
LIU Hui, LIU Zhangmin, FENG Yan, et al. Characterization of glutamine synthetase GLnA_Shy from Streptomyces[J].Journal of Nanjing Tech University(Natural Science Edition),2020,42(2):249-256..
更新日期/Last Update: 2020-03-20