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是未知的，即使是最好的研究模式生物，如谷氨酸棒杆菌，铜绿假单胞菌或沙门氏菌。使用纯化的蛋白质，我们最近已经表明，柠檬酸裂解酶β亚基样（CitE样）蛋白在鼠疫耶尔森菌和铜绿假单胞菌的功能作为（S）-citramalyl-CoA裂解酶衣康酸代谢。我们的研究结果揭示了衣康酸降解与致病性的联系，并表明衣康酸降解可以被视为各种病原体中的持久性因子。现在，我们想转移我们的衣康酸降解的体外模型获得的异源产生的Y。鼠疫杆菌的酶用于在含有衣康酸盐的基本培养基上生长的沙门氏菌细胞以及在感染期间的体内研究。此外，我们的初步数据表明，CitE家族的蛋白质的功能多样性：分支杆菌和盐细菌CitE催化（R）-3-羟基-3-甲基戊二酰辅酶A裂解酶反应的一个新的亮氨酸降解途径; C.谷氨酸CitE作为苹果酰辅酶A硫酯酶起作用，其它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）使用鼠伤寒沙门氏菌作为模型生物体在小鼠模型中进行体外和体内研究衣康酸降解对发病机制的重要性（与苏黎世的Wolf-Dietrich Hardt教授合作）;（ii）揭示新的亮氨酸降解途径的细节;（iii）检验C.谷氨酸CitE的代谢校正（与Michael Bott教授，Julich合作）;（iv）研究CitE蛋白在其他微生物中的功能，以及（v）进行结构研究，以了解这些酶底物特异性的分子基础（与Ulrich Ermler博士，法兰克福合作）。

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).