YfiD of Escherichia coli and Y061 of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase
YfiD of Escherichia coli and Y061 of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase
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DOI：
10.1006/bbrc.2001.5186
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发表时间：
2001-07-13
影响因子：
3.1
通讯作者：
Knappe, J
中科院分区：
文献类型：
--
作者：
Wagner, AFV;Schultz, S;Knappe, J
氧与丙酮酸甲酸裂解酶（Pn）中的甘氨酰自由基的反应导致Gly 734的N-C α之间的多肽骨架的裂解。此处显示包含Pn核心（Ser 1-Ser 733）的重组蛋白与大肠杆菌的YfiD蛋白（14 kDa）缔合，并且同样与同源T4编码的Y 06 I蛋白缔合，在与Pn活化酶反应时产生具有完全催化活性（35 U/nmol）的异源寡聚Pn酶。用氧处理活化的复合物导致14 kDa蛋白质裂解成11和3 kDa多肽，如对于推定的甘氨酰自由基在Gly 102（YfiD）或Gly 95（Y 06 I）处的定位所预期的。对于从Y 06 I分离的片段，质谱分析（nanoESI-MS）确定了11 kDa片段中的C-末端丝氨酸羧酰胺和3 kDa片段中的N-末端草酰修饰。推测E.大肠杆菌和其它兼性厌氧细菌已经进化为Pn的甘氨酰自由基结构域的“备用部分”，用于在经历氧化应激的细胞中快速恢复PFL活性并因此产生ATP。(C)北京：科学出版社.
Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (Pn) leads to cleavage of the polypeptide backbone between N-C alpha of Gly734. A recombinant protein comprising the core of Pn (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with Pn activase a heterooligomeric Pn enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for Pn's glycyl radical domain, utilized for rapid recovery of PFL activity land thus ATP generation) in cells that have experienced oxidative stress. (C) 2001 Academic Press.