Scholz Oliver, Köstner Martin, Reich Marco, Gastiger Susanne, Hillen Wolfgang
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
J Mol Biol. 2003 May 30;329(2):217-27. doi: 10.1016/s0022-2836(03)00427-3.
Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.
四环素阻遏蛋白（TetR）对诱导剂四环素（tc）具有高亲和力。tc类似物4-去（二甲基氨基）-6-脱氧-6-去甲基四环素（cmt3）不是TetR的诱导剂。通过定向进化方法分四个连续步骤筛选合适的TetR突变体，从而产生了对cmt3的诱导特异性。根据各自结合常数的比率判断，最佳TetR突变体对cmt3的特异性比对tc的特异性提高了20000多倍。通过DNA改组进行的两轮定向进化揭示了His64是诱导剂特异性的关键残基。具有cmt3特异性的最佳TetR突变体包含H64K交换，导致对tc的亲和力降低300倍，对cmt3的亲和力提高20倍。另一轮定向进化利用随机寡核苷酸对TetR的tc结合口袋附近的选定残基进行突变，得到了对cmt3亲和力提高250倍的TetR S135L。构建了双突变体TetR H64K S135L，并再次使用随机寡核苷酸进行定向进化，以改变tc结合口袋“二级壳层”中的残基。产生的最佳突变体TetR H64K E114Q S135L、TetR A61V H64K Q109E Q116E S135L和TetR H64K T112K S135L完全由cmt3诱导，而不由tc诱导。因此，它们的诱导剂特异性已被重新设计。基于与几种tc类似物的结合常数并根据TetR晶体结构，讨论了诱导剂识别改变的分子机制。
