The hydantoin transport protein from Microbacterium liquefaciens
The hydantoin transport protein from Microbacterium liquefaciens
复制标题
DOI：
10.1128/jb.188.9.3329-3336.2006
复制
发表时间：
2006-05-01
影响因子：
3.2
通讯作者：
Henderson, PJF
中科院分区：
文献类型：
--
作者：
Suzuki, S;Henderson, PJF
将来自微杆菌AJ 3912的基因hyuP与添加的His（6）标签一起克隆到大肠杆菌宿主菌株中的表达质粒pTTQ 18中。转化后的E.大肠杆菌显示运输放射性同位素标记的5-取代乙内酰脲的表观Km值在微摩尔范围内。该活性表现出最适pH值为6.6，并被二硝基苯酚抑制，表明运输系统的能量需求。5-吲哚基甲基乙内酰脲和5-苄基乙内酰脲是优选的底物，具有对乙内酰脲5位疏水取代基的选择性和对D异构体的L异构体的选择性。具有较小疏水性取代基的乙内酰脲，胞嘧啶，硫胺素，尿嘧啶，尿囊素，腺嘌呤和鸟嘌呤，不是有效的配体。His-标记的乙内酰脲转运蛋白位于内膜部分中，从中溶解并纯化，并鉴定其身份。
The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His(6) tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent K-m values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the L isomer over the D isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.