In vivo production of catalase containing haem analogues
In vivo production of catalase containing haem analogues
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DOI：
10.1111/j.1742-4658.2010.07677.x
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发表时间：
2010-06-01
期刊：
影响因子：
5.4
通讯作者：
Hederstedt, Lars
中科院分区：
文献类型：
--
作者：
Brugna, Myriam;Tasse, Lena;Hederstedt, Lars
血红素（原血红素IX）类似物是有毒化合物，并已被认为是用作抗菌剂，但其毒性背后的主要机制尚未得到证实。使用血红素蛋白过氧化氢酶在革兰氏阳性菌粪肠球菌作为一个实验系统，我们表明，各种血红素类似物可以被细菌细胞和纳入血红素依赖性酶。由此产生的辅因子取代的蛋白质是功能失调的，通常导致细胞生长停滞或死亡。这在很大程度上解释了血红素类似物的细胞毒性。与许多其他生物不同，E.粪肠球菌不依赖血红素生长，因此抵抗许多血红素类似物的毒性。我们利用这一特点，建立了一个细菌在体内系统的生产辅因子取代血红素蛋白的变体。作为试点研究，我们产生、分离和分析了新的过氧化氢酶变体，其中血红素辅基的铁原子被其他金属（即钴、镓、锡和锌）取代，以及含有中位血红素IX、钌中位原卟啉IX和（无金属）原卟啉IX的变体。这种类型的工程血红素蛋白在基础研究和生物技术工业中具有潜在用途。
Haem (protohaem IX) analogues are toxic compounds and have been considered for use as antibacterial agents, but the primary mechanism behind their toxicity has not been demonstrated. Using the haem protein catalase in the Gram-positive bacterium Enterococcus faecalis as an experimental system, we show that a variety of haem analogues can be taken up by bacterial cells and incorporated into haem-dependent enzymes. The resulting cofactor-substituted proteins are dysfunctional, generally resulting in arrested cell growth or death. This largely explains the cell toxicity of haem analogues. In contrast to many other organisms, E. faecalis does not depend on haem for growth, and therefore resists the toxicity of many haem analogues. We have exploited this feature to establish a bacterial in vivo system for the production of cofactor-substituted haem protein variants. As a pilot study, we produced, isolated and analysed novel catalase variants in which the iron atom of the haem prosthetic group is replaced by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants containing meso-protoheme IX, ruthenium meso-protoporphyrin IX and (metal-free) protoporphyrin IX. Engineered haem proteins of this type are of potential use within basic research and the biotechnical industry.