Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli
Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli
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DOI：
10.1080/09687860500092927
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发表时间：
2005-05-01
影响因子：
--
通讯作者：
Frillingos, S
中科院分区：
文献类型：
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作者：
Karatza, P;Frillingos, S
大肠杆菌K - 12基因组的编码潜能包括YgfO和YicE，它们是进化上保守的NAT/NCS2转运蛋白家族的两个成员，彼此高度同源（45%的残基同一性），并且与构巢曲霉的UapA密切相关，UapA是该家族中研究最为广泛的微生物成员。YgfO和YicE从基因组中克隆出来，在染色体外过量表达，并在大肠杆菌K - 12中相应内源性系统活性可忽略的条件下，检测其对[³H]黄嘌呤和其他碱基的摄取情况。通过用来自大肠杆菌乳糖通透酶的表位和来自肺炎克雷伯菌的生物素受体结构域进行C末端标记，构建了YgfO和YicE的替代的、基本等效的功能版本。结果表明，YgfO和YicE都存在于大肠杆菌的质膜中，并作为黄嘌呤的特异性、高亲和力转运蛋白发挥作用（YgfO的Km为4.2 - 4.6 μM，YicE的Km为2.9 - 3.8 μM），且依赖质子动力；在外部浓度高达0.1 mM时，它们对尿嘧啶、次黄嘌呤或尿酸没有可检测到的转运。YgfO和YicE在识别嘌呤环8位修饰的尿酸或黄嘌呤类似物（8 - 甲基黄嘌呤、8 - 氮杂黄嘌呤、氧嘌呤醇、别嘌呤醇）方面效率低下，这使它们有别于其真菌同源物UapA和Xut1。
The coding potential of the genome of E. coli K-12 includes YgfO and YicE, two members of the evolutionarily conserved NAT/NCS2 transporter family that are highly homologous to each other (45% residue identity) and closely related to UapA of Aspergillus nidulans, a most extensively studied microbial member of this family. YgfO and yicE were cloned from the genome, over-expressed extrachromosomally and assayed for uptake of [ 3 H] xanthine and other nucleobases, in E. coli K-12, under conditions of negligible activity of the corresponding endogenous systems. Alternative, essentially equivalent functional versions of YgfO and YicE were engineered by C-terminal tagging with an epitope from the E. coli lactose permease and a biotin-acceptor domain from Klebsiella pneumoniae. Both YgfO and YicE were shown to be present in the plasma membrane of E. coli and function as specific, high-affinity transporters for xanthine (K-m 4.2 - 4.6 mu M for YgfO, or 2.9 - 3.8 mu M for YicE), in a proton motive force-dependent manner; they display no detectable transport of uracil, hypoxanthine, or uric acid at external concentrations of up to 0.1 mM. Both YgfO and YicE are inefficient in recognizing uric acid or xanthine analogues modified at position 8 of the purine ring (8-methylxanthine, 8-azaxanthine, oxypurinol, allopurinol), which distinguishes them from their fungal homologues UapA and Xut1.