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-氧酸含量：硫氧还氧化还原酶（Ofors）的Sulfolobus sp。菌株7和来自气管的菌株用于制作X射线晶体学的晶体。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。从硫酸和其他来源的Ofors比较sulfolobus ofor中，可以选择识别2-氧的活性位点残基。和氨基酸对齐。为了促进这些残基的作用，它们受到了位置定向诱变，以获得认真的变体酶。亚基A的Arg344和Thr353分别是必不可少的和重要的。亚基-B的Lys49和Leu123分别负责识别2-氧基酸盐和丙酮酸酯。检查了一个应该与COA结合的GXXGXG基序。每种糖对于酶活性都是必不可少的，这表明基序的重要性。在研究过程中，已经发现了一种能够催化脱羧和吲哚吡式扭转酸酯氧化的酶。该反应与铁氧还蛋白或COA无关，其最终产物是吲哚乙酸。该酶是带有生物素的molybdo-flavo-ravo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。

福田英理子: "遺伝子の効率的な発現系を用いた好熱性古細菌由来の酸化還元酵素の機能解析"第23回日本分子生物学会年回プログラム・講演要旨集. 759 (2000)

Eriko Fukuda：“使用有效的基因表达系统对嗜热古菌衍生的氧化还原酶进行功能分析”第 23 届日本分子生物学会年度计划和摘要 759 (2000)。