All three residues of the Tn10 transposase DDE catalytic triad function in divalent metal ion binding
All three residues of the Tn10 transposase DDE catalytic triad function in divalent metal ion binding
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DOI：
10.1006/jmbi.1999.2837
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发表时间：
1999-06-25
影响因子：
5.6
通讯作者：
Haniford, DB
中科院分区：
文献类型：
--
作者：
Allingham, JS;Pribil, PA;Haniford, DB
Tn10/IS10转位涉及从供体位点切除转座子，然后将切除的转座子连接到新的靶位点。这些步骤是由tn10编码的转座酶蛋白催化的，需要合适的二价金属离子的存在。像其他转座酶和逆转录病毒整合酶蛋白一样，Tn10转座酶似乎包含一个单一的活性位点，其中包括一个通常称为DDE基序的酸性氨基酸残基三元组。除了催化作用外，Tn10转座酶DDE基序还在靶捕获中起作用，这一步骤在体外通过合适的二价金属离子的存在大大促进。我们发现，在Tn10转座酶的每个DDE基序残基上的半胱氨酸残基置换导致催化所需的二价金属离子发生变化，因此可以使用Mn2+而不能使用Mg2+。这种金属离子特异性的转换提供了证据，证明每个DDE基序残基直接在金属离子结合中起作用。我们还展示了DDE突变对金属离子辅助靶捕获的不同影响。许多模型，包括两个金属离子活性位点，被认为可以解释这些效应。(C) 1999学术出版社。
Tn10/IS10 transposition involves excision of the transposon from a donor site and subsequent joining of the excised transposon to a new target site. These steps are catalyzed by the Tn10-encoded transposase protein and require the presence of a suitable divalent metal ion. Like other transposase and retroviral integrase proteins, Tn10 transposase appears to contain a single active site which includes a triad of acidic amino acid residues generally referred to as the DDE motif. In addition to its role in catalysis, the Tn10 transposase DDE motif also functions in target capture, a step that in vitro is greatly facilitated by the presence of a suitable divalent metal ion. We show that cysteine residue substitutions at each of the DDE motif residues in Tn10 transposase result in a change in the divalent metal ion requirements for catalysis, such that Mn2+ but not Mg2+ can be used. This switch in metal ion specificity provides evidence that each of the DDE motif residues functions directly in metal ion binding. We also show differential effects of DDE mutations on metal ion-assisted target capture. A number of models, including a two metal ion active site, are considered to explain these effects. (C) 1999 Academic Press.