Baker T A, Luo L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6654-8. doi: 10.1073/pnas.91.14.6654.
A tetramer of Mu transposase (MuA) cleaves the phage Mu DNA and joins these ends to a target DNA to catalyze transposition. Substitution mutations at Asp-269 or Glu-392 within MuA destroy both the DNA cleavage and joining activities without blocking tetramer assembly, indicating that the mutations specifically affect catalysis. Although inactive under standard reaction conditions (10 mM Mg2+), the mutant proteins are partially resuscitated by 10-20 mM Mn2+, concentrations 5- to 10-fold higher than optimal for wild-type MuA. Amino acid sequence alignment and the similar effects of mutations suggests that Asp-269 and Glu-392 of MuA may be analogs of the first Asp and final Glu of a conserved triad of acidic amino acids present in many transposases and the retroviral integrases (the D-D-35-E motif). The higher Mn2+ optima observed with MuA derivatives altered at these positions supports a role for the conserved acidic amino acids in coordinating divalent metal ions in the active sites of transposases.
Mu转座酶（MuA）四聚体切割噬菌体Mu DNA，并将这些末端连接到靶DNA上以催化转座。MuA内天冬氨酸-269或谷氨酸-392处的取代突变会破坏DNA切割和连接活性，而不会阻止四聚体组装，这表明这些突变特异性地影响催化作用。尽管在标准反应条件（10 mM Mg2+）下无活性，但突变蛋白在10 - 20 mM Mn2+存在下可部分恢复活性，该浓度比野生型MuA的最佳浓度高5至10倍。氨基酸序列比对和突变的相似效应表明，MuA的天冬氨酸-269和谷氨酸-392可能类似于许多转座酶和逆转录病毒整合酶中存在的保守酸性氨基酸三联体的第一个天冬氨酸和最后一个谷氨酸（D-D-35-E基序）。在这些位置发生改变的MuA衍生物观察到较高的Mn2+最佳浓度，这支持了保守酸性氨基酸在转座酶活性位点中协调二价金属离子的作用。
