Functions and properties of citrate lyase beta-subunit-like proteins

基因组中编码的许多蛋白质的功能尚不清楚，即使是对谷氨酸棒杆菌、铜绿假单胞菌或肠沙门氏菌等研究最好的模式生物也是如此。利用纯化的蛋白质，我们最近证明了鼠疫耶尔森氏菌和铜绿假单胞菌中的柠檬酸裂解酶(CITE-like)蛋白在衣康酸代谢中起(S)-柠檬酸-辅酶A裂解酶的作用。我们的结果揭示了衣康酸降解与致病性之间的联系，并表明衣康酸降解可以被认为是各种病原体的持久性因素。现在，我们希望将我们用异源生产的鼠疫杆菌酶获得的衣康酸降解的体外模型转移到体内对沙门氏菌细胞的研究中，这些细胞生长在含有衣康酸的最低限度的培养基上，以及在感染期间。此外，我们的初步数据显示了CITE家族蛋白的功能多样性：分枝杆菌和盐杆菌CITE在一种新的亮氨酸降解途径中催化(R)-3-羟基-3-甲基戊二酰辅酶A裂解反应；C.Glamicum Cite具有malyl-CoA硫酯酶功能，其他CITE蛋白显然在代谢中具有不同的功能。在这个项目中，我们将(I)在体外和体内(与苏黎世Wolf-Dietrich Hardt教授合作)在小鼠模型中使用鼠伤寒沙门氏菌作为模型生物来研究衣康酸降解在发病机制中的重要性；(Ii)揭示一种新的亮氨酸降解途径的细节；(Iii)测试C.Glamicum Cite参与代谢校对的假设(与Michael Bott，Julich教授合作)；(4)研究CITE蛋白在其他微生物中的功能，以及(5)进行结构研究，以了解这些酶底物专一性的分子基础(与法兰克福的Ulrich Ermler博士合作)。

The function of many proteins encoded in the genomes is unknown even for best studied model organisms like Corynebacterium glutamicum, Pseudomonas aeruginosa or Salmonella enterica. Using purified proteins, we have recently shown that a citrate lyase ß-subunit-like (CitE-like) protein in Yersinia pestis and P. aeruginosa functions as (S)-citramalyl-CoA lyase in itaconate metabolism. Our results revealed the connection of itaconate degradation to pathogenicity and suggested that itaconate degradation can be regarded as a persistence factor in various pathogens. Now, we want to transfer our in vitro model of itaconate degradation obtained with heterologously produced Y. pestis enzymes into the in vivo studies of Salmonella cells grown on minimal media with itaconate as well as during infection. Furthermore, our preliminary data suggest functional diversity of proteins of CitE family: mycobacterial and halobacterial CitE catalyze (R)-3-hydroxy-3-methylglutaryl-CoA lyase reaction in a novel leucine degradation pathway; C. glutamicum CitE functions as malyl-CoA thioesterase, and other CitE proteins apparently have different functions in metabolism. In this project, we are going (i) to study the importance of itaconate degradation for pathogenesis using Salmonella enterica serovar Typhimurium as a model organism both in vitro and in vivo in a mouse model (in collaboration with Prof. Wolf-Dietrich Hardt, Zurich); (ii) to reveal details of a novel leucine degradation pathway; (iii) to test the hypothesis of the involvement of C. glutamicum CitE in metabolic proofreading (in collaboration with Prof. Michael Bott, Julich); (iv) to study functions of CitE proteins in other microorganisms, and (v) to perform structural studies in order to understand molecular bases for the substrate specificity of these enzymes (in collaboration with Dr. Ulrich Ermler, Frankfurt).