Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.
Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.
复制标题
在大肠杆菌转座子 Tn10 编码的金属四环素/H 逆向转运蛋白中，Ala-220 的第二位点突变为 Glu 或 Asp，抑制了 Asp-285 突变为 Asn。
DOI：
10.1016/s0021-9258(19)74208-2
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
T. Sawai
中科院分区：
文献类型：
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作者：
A. Yamaguchi;R. O'yauchi;Y. Someya;T. Akasaka;T. Sawai
Asp-285的羧基对于由大肠杆菌的转座子Tn 10编码的金属-四环素/H+反向转运蛋白（TetA）介导的四环素/H+反向转运蛋白是必需的（Yamaguchi，A.，赤坂，T.，Ono，N.，Someya，Y.，Nakatani，M.，和Sawai，T.（1992）J.Biol.Chem.267，7490-7498）。从大肠杆菌中分离到自发性四环素抗性回复突变体。大肠杆菌细胞携带Asn-285突变tetA基因。所有的回复突变体都是由于GCG（Ala）第二位密码子220突变为GAG（Glu）所致。由回复突变体TetA蛋白介导的四环素转运的Km值比野生型高约4倍，表明回复突变体是低亲和力突变体。通过定点突变构建的Glu-220和Asn-285双突变体表现出与回复突变体相同的性质，证实Ala-220的突变是唯一负责抑制的。Asn-285突变体的Asp-220突变导致四环素抗性和转运活性的恢复水平低于Glu-220突变的情况。用Glu或Asp取代Ala-220的单个突变导致四环素抗性降低约2-4倍，但转运活性没有关键变化。电荷中和盐桥不太可能需要Glu-220，因为Glu-220或Asp-220单突变体中的未配对负电荷不会引起活性的严重变化。另一种解释是合理的; Asp-285直接有助于阳离子底物、金属-四环素螯合复合物或质子的结合，220位的酸性残基可以取代Asp-285的作用。
A carboxyl group of Asp-285 is essential for tetracycline/H+ antiport mediated by the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA) of Escherichia coli (Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M., and Sawai, T. (1992) J. Biol. Chem. 267, 7490-7498). Spontaneous tetracycline resistance revertants were isolated from E. coli cells carrying the Asn-285 mutant tetA gene. All of the revertants were due to the second-site mutation at codon 220 of GCG (Ala) to GAG (Glu). The Km value of the tetracycline transport mediated by the revertant TetA protein was about 4-fold higher than that of the wild-type, indicating that the revertant is a low affinity mutant. A Glu-220 and Asn-285 double mutant constructed by site-directed mutagenesis showed the same properties as the revertants, confirming that the mutation of Ala-220 is solely responsible for the suppression. The Asp-220 mutation of the Asn-285 mutant resulted in a lower level of restoration of the tetracycline resistance and the transport activity than in the case of the Glu-220 mutation. A single mutation replacing Ala-220 with Glu or Asp caused about a 2-4-fold decrease in the tetracycline resistance, but no crucial change in the transport activity. It is not likely that Glu-220 is required for a charge-neutralizing salt bridge because an unpaired negative charge in a Glu-220 or Asp-220 single mutant did not cause a serious change in the activity. An alternative explanation is reasonable; Asp-285 directly contributes to the binding of a cationic substrate, metal-tetracycline chelation complex, or proton, and an acidic residue at position 220 can take over the role of Asp-285.