摘要
CrgA已经被证实是三孢布拉霉和一些其他丝状真菌中类胡萝卜素生物合成的一个负调控因子。环指结构域的存在表明,CrgA可能具有泛素连接酶的功能,是泛素化调节系统中的一个关键酶。为了验证这一假设,从三孢布拉霉中分离并鉴定了一种泛素激活酶(BtE1)和18种假定的泛素结合酶(UBC)。生物信息学分析表明,18种UBC蛋白质都包含一个UBC的保守结构域,表明它们都属于泛素结合酶。系统发育关系分析表明,18个UBC蛋白质属于7个蛋白质亚家族。根据系统进化分析结果筛选出6种候选UBC蛋白质,通过异源表达及亲和纯化得到BtE1、6种BtUBC蛋白质、BtCrgA和BtWC-1b,并在体外进行泛素化反应,BtCrgA能在BtE1和BtUBC D的协助下完成对自身的泛素修饰功能。
CrgA has been identified as a negative regulator of the carotenoid biosynthesis in Blakeslea trispora and some other zygomycetes.The presence of the ring finger domains suggest that CrgA may function as an ubiquitin ligase,a key enzyme in the ubiquitination regulation system.In order to test this hypothesis,one ubiquitin activating enzyme(BtE1)and 18 putative ubiquitin-conjugation enzymes(UBC)from B.trispora were isolated and identified.Bioinformatics analysis indicated that each of the 18 UBC proteins contained a conserved domain of UBC,indicating that all of them belong to ubiquitin binding enzymes.Phylogenetic relationship analysis showed that 18 UBC proteins belong to 7 protein subfamilies,homologous with those of Saccharomyces cerevisiae.6 candidate UBC proteins were screened based on phylogenetic analysis,and BtE1,6 BtUBC proteins,BtCrgA and BtWC-1b were obtained by heterologous expression and affinity purification,which were ubiquitinated in vitro.BtCrgA can perform ubiquitin modification function on itself with the assistance of BtE1 and BtUBC D.
作者
杨佳敏
杨培龙
曲音波
余晓斌
罗玮
YANG Jiamin;YANG Peilong;QU Yinbo;YU Xiaobin;LUO Wei(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Feed Biotechnology,Ministry of Agriculture and Rural Affairs of People's Republic of China,Beijing 100008,China;Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100008,China;State Key Laboratory of Microbial Technology,Shandong University,Qingdao 266237,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2024年第2期81-88,共8页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(21878123)
农业农村部饲料生物技术重点实验室开放课题(KLFB-FRI-202001)
微生物技术国家重点实验室开放课题(M2020-06)。
关键词
CrgA
泛素连接酶
泛素活化酶
泛素结合酶
体外泛素化
CrgA
ubiquitin ligase
ubiquitin activating enzyme
ubiquitin binding enzyme
in vitro ubiquitination reaction
