Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.
Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.
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静电抑制允许在不存在保守的催化精氨酸的情况下进行酪氨酸位点特异性重组。
DOI：
10.1074/jbc.m110.112292
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Jayaram,Makkuni
中科院分区：
文献类型：
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作者：
Rowley,PaulA;Kachroo,AashiqH;Ma,Chien-Hui;Maciaszek,AnnaD;Guga,Piotr;Jayaram,Makkuni
酪氨酸家族位点特异性重组酶Flp的活性位点含有保守的催化五联体，其包括两个精氨酸残基，Arg-191和Arg-308。这两种精氨酸都是在易切割位置含有磷酸基团的DNA底物中链切割和链连接的酯交换步骤所必需的。在链裂解过程中，活性位点酪氨酸提供亲核试剂，形成共价3′-磷酸酪氨酰中间体。通过裂解产生的5′-羟基提供亲核试剂以在重组DNA链中重新形成3′-5′磷酸二酯键。在以前的工作中，我们表明，易断裂的磷酸（P）的电荷中性甲基膦酸酯（MeP）的取代，使精氨酸-308在催化活化的MeP二酯键。然而，在Flp（R308 A）反应中，水与酪氨酸亲核试剂（Tyr-343）竞争，导致MeP二酯键的直接水解。我们现在报告，MeP激活也不需要Arg-191。与Flp（R308 A）相反，Flp（R191 A）主要介导Tyr-343的正常切割，但也表现出较弱的直接水解活性。由Flp（R191 A）形成的裂解的MeP-酪氨酰中间体可以被5′-羟基或水靶向进行亲核攻击，并分别引导至链连接或水解。与野生型Flp合作，Flp（R191 A）促进MeP-和P-DNA伴侣之间的链交换。因此，通过在DNA底物中进行补偿性修饰，中和易断裂的磷酸盐上的负电荷，可以抑制催化关键的带正电荷的侧链的损失。
The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.