Studies on a hyperthermostable 4-α-glucanotransferase: X-ray structure analysis and enzymatic reaction mechanism

超热稳定4-α-葡聚糖转移酶的研究:X射线结构分析和酶反应机制

基本信息

项目摘要

4-α-Glucanotransferase (Gtase) of a hyperthermophilic archaeon, Thermococcus litoralis, synthesizes linear and cyclic α-I ,4-glucans from oligomaltose and amylose as substrates. Following results were obtained in this research.(1) The Gtase gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the gene in E. coli resulted in low production of the enzyme and the accumulation of inclusion bodies. However, simultaneous expression of Gtase with tRNAィイD2AGAィエD2, tRNAィイD2AGGィエD2 and GroELS affected both the production and solubility of the enzyme, and production of soluble Gtase increased about 5-fold.(2) This enzyme reaction was carried out through the Ping-Pong BiBi mechanism, when analyzed with substrates, 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (donor) and glucose (acceptor).(3) This Gtase belongs to the family 57 of glycosyl hydrolases. No active site residues are identified in the family 57 enzymes. Comparing the amino acid sequences, 8 carboxylic resides were conserved in the family. Analyses of the mutant enzymes constructed by site-directed mutagenesis suggested that Gln 123, Asp 145, Asp2l4, Glu216, and Asp354 are present in or around the active site.(4) Crystals of the enzyme were obtained by using ammonium sulfate and PEG400 as precipitants. The space group was P6ィイD24ィエD222, and the unit cell dimensions were a=b= 125 Å and c=246 Å.
嗜热古菌滨海热球菌(Thermococcus litoralis)的4-α-葡聚糖转移酶(Gtase)以低聚麦芽糖和直链淀粉为底物合成直链和环状α-I,4-葡聚糖。本研究取得了以下成果。(1)Gtase基因具有高含量的精氨酸的阿加和AGG密码子,这在大肠杆菌中是极其罕见的。在E.大肠杆菌中,导致酶的产量低和包涵体的积累。然而,同时表达Gtase与tRNA内切酶D2 AGA内切酶D2、tRNA内切酶D2 AGG内切酶D2和GroELS影响酶的产生和溶解性,并且可溶性Gtase的产生增加约5倍。(2)当以3-酮亚丁基-β-2-氯-4-硝基苯基-麦芽五糖苷(供体)和葡萄糖(受体)为底物时,该酶反应通过乒乓BiBi机制进行。(3)该Gtase属于糖基水解酶家族57。在家族57酶中未鉴定出活性位点残基。氨基酸序列分析表明,该家族有8个保守的羧基残基。通过定点诱变构建的突变酶的分析表明,Gln 123,Asp 145,Asp 214,Glu 216和Asp 354存在于活性位点中或其周围。(4)以硫酸铵和PEG 400为沉淀剂,得到了酶的晶体。空间群为P6 D24 D222,晶胞尺寸为a=B= 125,c=246。

项目成果

期刊论文数量(11)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
伏信進矢ほか: "Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase biased distribution of acidic residues and importance of Asp37 for catalysis at low pH." Protein Engineering.11(12). 1121-1128 (1998)
Shinya Fushinobu 等人:“酸性残基的极端嗜酸和酸稳定木聚糖酶偏向分布的晶体学和突变分析以及 Asp37 在低 pH 条件下催化的重要性。11(12) (1998)。”
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田中照道ほか: "Engineering of S2 site of aqualysin I;alteration of P2 specificity by excluding P2 side chain." Biochemistry. 37(50). 17402-17407 (1998)
Terumichi Tanaka 等人:“aqualysin I 的 S2 位点工程;通过排除 P2 侧链改变 P2 特异性”37(50)。
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若木高善 ほか: "Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon Sulfolobus sp.strain 7 and overproduction of the enzyme by coexpression of tRNA for arginine rate codon." Biosci.Biotechnol.Biochem.62(12). 2408-2414 (1998)
Takayoshi Wakagi 等人:“从嗜热嗜酸古菌 Sulfolobus sp.strain 7 中克隆无机焦磷酸酶基因,并通过精氨酸速率密码子的 tRNA 共表达来过量生产该酶。Biosci.Biotechnol.Biochem.62(12)。” - 2414 (1998)
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Imamura, Hiromi; Jeon, Beong-Sam; Wakagi, Takayoshi; and Matsuzawa, Hiroshi: "High level expression of Thermococcus litoralis 4-α-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and Gr
Imamura,Hiromi;Jeon,Beong-Sam;Takayoshi 和 Matsuzawa,Hiroshi:“在大肠杆菌中以可溶形式高水平表达嗜热球菌 4-α-葡聚糖转移酶,涉及少量精氨酸 tRNA 和 Gr
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小関卓也ほか: "Purification and characterization of aferuloylesterase from Aspergillus awamori." Biosci.Biotechnol.Biochem.62(10). 2032-2034 (1998)
Takuy​​a Koseki 等人:“泡盛曲霉的阿魏酸酯酶的纯化和表征。Biosci.Biotechnol.Biochem.62(10) (1998)。
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MATSUZAWA Hiroshi其他文献

MATSUZAWA Hiroshi的其他文献

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{{ truncateString('MATSUZAWA Hiroshi', 18)}}的其他基金

On a study of a solution with a transition layer for a bistable reaction diffusion equation with a heterogeneous environment
非均相环境双稳态反应扩散方程带过渡层解的研究
  • 批准号:
    20740098
  • 财政年份:
    2008
  • 资助金额:
    $ 8.83万
  • 项目类别:
    Grant-in-Aid for Young Scientists (B)
Analyses of the crystal structure of 4-α-glucanotransferase and its production mechanism of new cycloamylose
4-α-葡聚糖转移酶晶体结构分析及其新型环直链淀粉生产机制
  • 批准号:
    12460047
  • 财政年份:
    2000
  • 资助金额:
    $ 8.83万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Analyzes of the mechanisms of thermophilic and alkalophilic properties of aqualysin I,a protease from an extreme thermophile
极端嗜热菌蛋白酶 aqualysin I 的嗜热和嗜碱特性机制分析
  • 批准号:
    08456046
  • 财政年份:
    1996
  • 资助金额:
    $ 8.83万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Structure and function of penicillin-binding protein from methicillinresistans Staphylococcus aureus
耐甲氧西林金黄色葡萄球菌青霉素结合蛋白的结构和功能
  • 批准号:
    06454074
  • 财政年份:
    1994
  • 资助金额:
    $ 8.83万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (B)
Study on Extracelluar Secretion and Folding Mechanism of a Thermophilic Protease (Aqualysin I)
嗜热蛋白酶(Aqualysin I)胞外分泌及折叠机制的研究
  • 批准号:
    02454060
  • 财政年份:
    1990
  • 资助金额:
    $ 8.83万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (B)
Structure and Function of the RodA Protein Responsible for the Rod Shape of Escherichia coli
负责大肠杆菌杆状形状的 RodA 蛋白的结构和功能
  • 批准号:
    62560072
  • 财政年份:
    1987
  • 资助金额:
    $ 8.83万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)

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La 和 La 相关蛋白对基因表达的调节
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