Two ATP-binding cassette transporters involved in (S)-2-aminoethyl-cysteine uptake in Thermus thermophilus
Two ATP-binding cassette transporters involved in (S)-2-aminoethyl-cysteine uptake in Thermus thermophilus
复制标题
两个 ATP 结合盒转运蛋白参与嗜热栖热菌 (S)-2-氨乙基-半胱氨酸的摄取
DOI：
10.1128/jb.00202-13
复制
发表时间：
2013
影响因子：
3.2
通讯作者：
M. Nishiyama
中科院分区：
文献类型：
--
作者：
Y. Kanemaru;F. Hasebe;T. Tomita;T. Kuzuyama;M. Nishiyama
嗜热栖热菌对赖氨酸类似物（S）-2-氨乙基-半胱氨酸（AEC）表现出超敏反应。使用来自两个AEC抗性突变体AT 10和AT 14的基因组构建粘粒文库，并分离赋予野生型菌株AEC抗性的粘粒。当对突变体AT 14的粘粒文库进行筛选时，获得了两个独立的cosplay，赋予野生型部分AEC抗性。两个互补序列携带一个共同的基因组区域，从TTC 0795到TTC 0810。该区域含有编码ATP结合盒（ABC）转运蛋白的基因，该转运蛋白由TTC 0806/TTC 0795组成，使用TTC 0807作为周质底物结合蛋白。测序结果表明，AT 14基因在TTC 0969和TTC 0795位点发生突变，导致产物的热稳定性降低，TTC 0969编码一个不同ABC转运蛋白的核苷酸结合蛋白，该蛋白由TTC 0966作为周质底物结合蛋白的TTC 0967/TTC 0968/TTC 0969/TTC 0970组成。通过对突变体AT 10构建的互补链进行类似的筛选，发现突变位于TTC 0807和TTC 0969。突变的转运蛋白组分中的任一个在野生型菌株中对AEC产生部分抗性，而两种转运蛋白的突变赋予完全的AEC抗性。这一结果表明，这两种转运蛋白都参与了T.嗜热菌为了阐明AEC摄取的机制，以几种底物结合形式测定了TTC 0807的晶体结构。结构显示，TTC 0807通过改变Glu 19的侧链构象识别各种碱性氨基酸，Glu 19与底物的侧链氨基相互作用。
Thermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids, conferring partial AEC resistance to the wild type, were obtained. Two cosmids carried a common genomic region fromTTC0795toTTC0810. This region contains genes encoding an ATP-binding cassette (ABC) transporter consisting of TTC0806/TTC0795, using TTC0807 as the periplasmic substrate-binding protein. Sequencing revealed that AT14 carries mutations inTTC0795andTTC0969, causing decreases in the thermostability of the products.TTC0969encodes the nucleotide-binding protein of a different ABC transporter consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0966 as the periplasmic substrate-binding protein. By similar screening for cosmids constructed for the mutant AT10, mutations were found atTTC0807andTTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substrate-binding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.