Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon Tn10. Histidine 257 plays an essential role in H+ translocation.
Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon Tn10. Histidine 257 plays an essential role in H+ translocation.
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DOI：
10.1016/s0021-9258(18)38081-5
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发表时间：
1991-04
期刊：
影响因子：
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通讯作者：
Akihito Yamaguchi;Kumiko Adachi;Takaaki Akasaka;Nobukazu Ono;Tetsuo Sawai
中科院分区：
文献类型：
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作者：
Akihito Yamaguchi;Kumiko Adachi;Takaaki Akasaka;Nobukazu Ono;Tetsuo Sawai
转座子Tn10编码的TETA基因产物是一个金属-四环素/质子逆向转运蛋白(Yamaguchi，A.，Udagawa，T.和Sawai，T.(1990)J.Biol)。化学。265、4809-4813)。其四环素转运活性可被组氨酸专一性试剂焦碳酸二乙酯抑制。在这个反向转运蛋白的5个组氨酸残基中，只有His257位于可能的跨膜螺旋上。因此，His257被Glu或Asp取代。含有Glu257和Asp257突变蛋白的倒置囊泡对四环素的摄取分别只有野生型囊泡的20%和10%。与野生型囊泡相比，突变型囊泡没有四环素依赖的质子转运，表明突变蛋白失去了四环素/H+逆向转运活性。突变囊泡对~(60)Co~(2+)的显著吸收而没有质子转移，也证实了突变载体是金属-四环素复合体的单一转运体。突变蛋白对金属四环素的单一转运不受焦碳酸二乙酯的抑制，表明His257是质子转运所必需的唯一组氨酸残基。这些突变蛋白对四环素产生了大约一半的抗药性，可能是因为它们催化金属-四环素复合体下坡流出细胞。
The transposon Tn10-encoded tetA gene product is a metal-tetracycline/proton antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). Its tetracycline transport activity was inhibited by a histidine-specific reagent, diethyl pyrocarbonate. Among five histidine residues in this antiporter, only His257 is located in the putative transmembrane helices. Thus, His257 was replaced by Glu or Asp. Inverted vesicles containing the Glu257 and Asp257 mutant proteins showed only 20 and 10% of the tetracycline uptake of wild-type vesicles, respectively. In contrast to wild-type vesicles, the mutant vesicles showed no tetracycline-dependent proton translocation, indicating that the mutant proteins had lost the tetracycline/H+ antiport activity. The significant 60Co2+ uptake without proton translocation by the mutant vesicles also confirmed that the mutant carriers act as uniporters of a metal-tetracycline complex. The metal-tetracycline uniport by the mutant proteins was not inhibited by diethyl pyrocarbonate, indicating that His257 is the only histidine residue essential for proton translocation. These mutant proteins conferred about half-level resistance to tetracycline, probably due to their catalyzing downhill efflux of a metal-tetracycline complex out of the cells.