Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport.
Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport.
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发表时间：
1990-09
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通讯作者：
Akihito Yamaguchi;Nobukazu Ono;Takaaki Akasaka;T. Noumi;Tetsuo Sawai
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作者：
Akihito Yamaguchi;Nobukazu Ono;Takaaki Akasaka;T. Noumi;Tetsuo Sawai
转座子Tn10编码的四环素抗性蛋白具有金属-四环素/H+逆向转运蛋白的功能(Yamaguchi，A.，Udagawa，T.和Sawai，T.(1990)J.Biol)。化学。265、4809-4813)。Ser65-Asp66二肽在所有已知的四环素逆向转运蛋白中都是保守的，是定点突变的重要靶点。当Asp66被Asn取代时，转运活性完全丧失，而当它被Glu取代时，活性降低到野生型水平的10%，表明66位的负电荷是四环素转运所必需的。相反，用半胱氨酸或丙氨酸取代Ser65只引起四环素转运活性的微小变化。然而，突变的Cys65反向转运蛋白对巯基试剂敏感。底物不能阻止N-乙基马来酰亚胺对Cys65逆向转运体的完全失活。一种体积较小的试剂，甲硫磺酸甲酯，在不改变其与底物的亲和力的情况下，导致Cys65逆向转运体部分失活。这些结果表明，除了底物结合外，含有该二肽的区域在金属-四环素的转运中起着重要作用。它可以作为一扇门，打开Asp66与金属四环素之间的电荷相互作用。
The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.