Crystal structure and functional analysis of 2-oxacid : ferredoxin oxidoreductase

2-含氧酸的晶体结构和功能分析：铁氧还蛋白氧化还原酶

• 批准号: 12660067
• 负责人: WAKAGI Takayoshi
• 金额: $ 2.3万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660067/
• 关键词: 2-oxoacid; ferredoxin; oxidoreductase; substrate specificity; thermophile; archaea; Sulfolobus; Aeropyrum; 耐熱性酵素

2-Oxoacid : ferredoxin oxidoreductases (OFORs) from Sulfolobus sp. Strain 7 and from Aeropyrum were used for making crystals for X-ray crystallography. However, some of them formed crystals which were found to diffract X-ray with very low resolution.Five possible active site residues that recognize 2-oxoacid in Sulfolobus OFOR were selected from the comparison of OFORs from Sulfolobus and other sources in terms of teriary structure and amino acid alignment. To eleuidate the role of these residues, they were subjected to site-directed mutagenesis to obtain a serious of variant enzymes. Arg344 and Thr353 of the subunit-a were essential and important, respectively. Lys49 and Leu123 of the subunit-b were responsible for the recognition of 2-oxoglutarate and pyruvate, respectively. A GXXGXG motif, supposed to bind to CoA, was examined. Each of the Gly was essential for enzyme activity, suggesting the importance of the motif.During the course of OFOR research, an enzyme capable of catalyzing both decarboxylation and oxidation of indolepyruvate has been discovered. The reaction was independent of ferredoxin or CoA, and its final product was indoleacetic acid. The enzyme was a molybdo-flavo-iron-sulfur protein with biotin.

2-含氧酸：来自硫化叶菌属的铁氧还蛋白氧化还原酶（OFOR）。来自 Aeropyrum 的菌株 7 用于制备 X 射线晶体学晶体。然而，其中一些形成的晶体被发现以非常低的分辨率衍射X射线。通过对来自Sulfolobus和其他来源的OFOR的三级结构和氨基酸排列进行比较，选择了识别Sulfolobus OFOR中2-含氧酸的五个可能的活性位点残基。为了阐明这些残基的作用，对它们进行定点诱变以获得一系列变异酶。 a亚基的Arg344和Thr353分别是必需的和重要的。 b 亚基的 Lys49 和 Leu123 分别负责识别 2-酮戊二酸和丙酮酸。检查了应该与 CoA 结合的 GXXGXG 基序。每个甘氨酸对于酶活性都是必需的，这表明该基序的重要性。在 OFOR 研究过程中，发现了一种能够催化吲哚丙酮酸脱羧和氧化的酶。该反应不依赖于铁氧还蛋白或CoA，其最终产物是吲哚乙酸。该酶是一种含有生物素的钼黄铁硫蛋白。

项目成果

西澤, 福田, 若木: "Aeropyrum pernixの二つの2-オキソ酸:フェレドキシン酸化還元酵素"日本農芸化学会誌. 75. 314 (2001)

Nishizawa、Fukuda、Wakagi：“两种 2-含氧酸：Aeropyrum pernix 中的铁氧还蛋白氧化还原酶”日本农业化学学会杂志 75. 314 (2001)。

E. Fukuda, T. Wakagi: "Substrate recognition by 2-oxoacid : ferredoxin oxidoreductase from Sulfolobus sp. strain 7"Biochim. Biophys. Acta. (in press).

E. Fukuda，T. Wakagi：“2-含氧酸的底物识别：来自硫化叶菌属菌株 7 的铁氧还蛋白氧化还原酶”Biochim。

福田, 若木: "好酸好熱性古細菌Sulfolobus sp. 7の2-オキソ酸:フェレドキシン酸化還元酵素(OFOR)の2-オキソ酸認識部位"日本農芸化学会誌. 75. 314 (2001)

Fukuda, Wakagi：“铁氧还蛋白氧化还原酶 (OFOR) 的 2-含氧酸识别位点：嗜酸古菌 Sulfolobus sp. 7 中的 2-含氧酸” 日本农业化学学会杂志 75. 314 (2001)。

E.Fukuda, H.Kino, H.Matsuzawa, T.Wakagi: "Role of a highly conserved YPITP motif in 2-oxoacid : ferredoxin oxidoreductase"Eur. J. Biochem.. 268. 5639-5646 (2001)

E.Fukuda、H.Kino、H.Matsuzawa、T.Wakagi：“2-含氧酸中高度保守的 YPITP 基序的作用：铁氧还蛋白氧化还原酶”Eur。

T.Wakagi, E.Fukuda, Y.Ogawa, H.Kino, H.Matsuzawa: "A novel bifunctional molybdo-enzyme catalyzing both decarboxylation of indolepyruvate and oxidation of indoleacetaldehyde from a thermoacido philic archaeon, Sulfolobus sp. strain 7"FEBS Letters. 510. 196

T.Wakagi、E.Fukuda、Y.Okawa、H.Kino、H.Matsuzawa：“一种新型双功能钼酶，可催化嗜热嗜酸古菌 Sulfolobus sp. 菌株 7 中吲哚丙酮酸的脱羧和吲哚乙醛的氧化”FEBS Letters