Steiner Amanda, Raheem Samah, Ahmad Zulfiqar
Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO 63501, USA.
Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO 63501, USA.
Int J Biol Macromol. 2020 Dec 15;165(Pt B):2588-2597. doi: 10.1016/j.ijbiomac.2020.10.133. Epub 2020 Oct 23.
The current study investigated the significance of βLeu-382 and βSer-383 residues in the highly conserved βDELSEED-loop of Escherichia coli ATP synthase. E. coli wild type and mutant enzymes were inhibited by the honeybee venom peptide melittin, which is a known ATP synthase inhibitor. The wild type enzyme was fully inhibited by melittin. Melittin-induced inhibitory profiles of single mutants βL382A/R/Q/D/E and βS383A/R/Q/D/E followed the pattern of wild-type enzymes with 7% to 30% residual activity. Double mutants βL382A/βS383A, βL382E/βS383E, and βL382R/βS383R retained 30%, 80%, and 78% residual activity, respectively. Variable loss of oxidative phosphorylation was observed in mutant enzymes, which was also reflected in the comparative growth of wild type and mutant E. coli strains. Double mutant enzymes βL382E/βS383E and βL382R/βS383R showed significant resistance to the melittin-induced inhibition. Wild type and mutant E. coli strains showed variable loss of growth in the presence of melittin. Indicial growth loss of E. coli strains and inhibition of isolated ATP synthase suggested that βLeu-382 and βSer-383 are vital for the function of enzyme. Individual loss of βLeu-382 and βSer-383 does not affect the melittin-induced inhibition. However, loss of both βLeu-382 and βSer-383 obstructs the inhibition suggesting loss of peptide binding at the βDELSEED-loop of ATP synthase.
本研究调查了大肠杆菌ATP合酶高度保守的βDELSEED环中βLeu - 382和βSer - 383残基的重要性。大肠杆菌野生型和突变型酶受到蜜蜂毒液肽蜂毒明肽的抑制，蜂毒明肽是一种已知的ATP合酶抑制剂。野生型酶被蜂毒明肽完全抑制。单突变体βL382A/R/Q/D/E和βS383A/R/Q/D/E的蜂毒明肽诱导抑制谱遵循野生型酶的模式，残留活性为7%至30%。双突变体βL382A/βS383A、βL382E/βS383E和βL382R/βS383R分别保留了30%、80%和78%的残留活性。在突变型酶中观察到氧化磷酸化的可变损失，这也反映在野生型和突变型大肠杆菌菌株的比较生长中。双突变体酶βL382E/βS383E和βL382R/βS383R对蜂毒明肽诱导的抑制表现出显著抗性。野生型和突变型大肠杆菌菌株在蜂毒明肽存在下表现出可变的生长损失。大肠杆菌菌株的生长损失和分离的ATP合酶的抑制表明βLeu - 382和βSer - 3 eighty-three对酶的功能至关重要。βLeu - 382和βSer - 383的单独缺失不影响蜂毒明肽诱导的抑制。然而，βLeu - 382和βSer - 383两者的缺失阻碍了抑制，表明ATP合酶βDELSEED环处的肽结合丧失。
