Characterisation of haem lyases and their dedicated roles in bacterial cytochrome c biogenesis

背景：C型细胞色素是一类广泛存在的蛋白质，在包括多种病原体在内的原核生物和真核生物的能量守恒代谢中起着至关重要的作用。细胞色素c生物发生的关键特征是血红素辅因子与排列在血红素c结合基序中的两个(或少数情况下仅一个)半胱氨酸残基共价结合，典型的是CXXCH。这个反应是由膜结合酶血红素裂解酶催化的。细菌血红蛋白裂解酶目前还没有得到纯化，其反应机理也不清楚。已经描述了两个独立的催化细菌细胞色素c生物合成的酶系统，但只对大肠杆菌的CCM系统(或系统I)进行了详细的研究。目标：本项目旨在揭示血球裂解酶在CCSA系统(或系统II)中的功能。可以说，最适合的模式细菌是产琥珀酸狼菌。它的基因组编码三种不同的血红素裂解酶，预计它们对不同的血红素C结合基序的特异性不同。这将检查野生型和血红素酶基因缺失突变体是否能够将血红素共价结合到包含经典或各种非传统的血红素C结合基序的模型c-型细胞色素上。一个血红素裂解酶(NrfI)已经被证明识别五氢细胞色素c亚硝酸盐还原酶的一个独特的CXXCK血红蛋白C结合基序。NRFI将进行定点修饰，以确定必需的氨基酸残基。将尝试对系统II的溶血酶进行过量生产、纯化和生化特性的研究。影响：拟议的工作将拓宽翻译后蛋白质成熟和预测Hem C结合基序的一般知识。此外，它的目标是建立产琥珀酸假单胞菌作为高效生产c型细胞色素的宿主。关键词：细胞色素c成熟；细胞色素c裂解酶；血红素c结合基序；产琥珀酸狼疮杆菌；细菌能量代谢。

Background: C-type cytochromes are a widespread class of proteins that play a vital role in the energy-conserving metabolism of prokaryotic and eukaryotic organisms including many pathogens. The key feature in cytochrome c biogenesis is the covalent attachment of the haem cofactor to two (or rarely only one) cysteine residues arranged in a haem c binding motif, typically CXXCH. This reaction is catalysed by the membranebound enzyme haem lyase. No bacterial haem lyase has ever been purified and its reaction mechanism is unknown. Two independent enzymic systems catalysing bacterial cytochrome c biogenesis have been described but only the Ccm system (or system I) of Escherichia coli has been investigated in any detail. Goals: This project aims to reveal the function of haem lyases involved in the CcsA system (or system II). The arguably best-suited model bacterium is Wolinella succinogenes. Its genome encodes three distinct haem lyases that are expected to differ in their specificity towards distinct haem c binding motifs. It will be examined whether or not W. succinogenes wild-type and haem lyase gene deletion mutants are able to covalently attach haem to a model c-type cytochrome containing either the classical or various unconventional haem c binding motifs. One haem lyase (NrfI) has already been shown to recognise a unique CXXCK haem c binding motif of pentahaem cytochrome c nitrite reductase. NrfI will be subject to site-directed modification in order to identify essential amino acid residues. Overproduction, purification and biochemical characterisation of system II haem lyases will be attempted. Impact: The proposed work will broaden the general knowledge on posttranslational protein maturation and prediction of haem c binding motifs. Furthermore, it aims to establish W. succinogenes as host for efficient c-type cytochrome production. Key words: Cytochrome c maturation; Cytochrome c haem lyase (CCHL); Haem c binding motif; Wolinella succinogenes; Bacterial energy metabolism.