Catalytic mechanism of cyclic Di-GMP-specific phosphodiesterase:: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa
Catalytic mechanism of cyclic Di-GMP-specific phosphodiesterase:: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa
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DOI：
10.1128/jb.00165-08
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发表时间：
2008-05-01
影响因子：
3.2
通讯作者：
Liang, Zhao-Xun
中科院分区：
文献类型：
--
作者：
Rao, Feng;Yang, Ye;Liang, Zhao-Xun
EAL结构域蛋白是维持细菌中第二信使环二GMP细胞浓度的主要磷酸二酯酶。鉴于EAL结构域在调节许多细菌行为中的关键作用，阐明其催化和调节机制将有助于破译环二GMP信号网络的努力。在这里，我们提出的数据表明，RocR，EAL结构域蛋白，调节铜绿假单胞菌PAO-1的毒力基因的表达和生物膜的形成，催化环二GMP的水解，通过使用一般的碱催化机制与镁离子的援助。除了参与Mg 2+结合的五个必需残基外，我们建议必需残基E。作为一般的碱催化剂，帮助Mg 2+配位的水去质子化，产生亲核的氢氧根离子。其他保守残基的突变引起不同程度的变化，在k（猫）或K-m，使我们提出他们的残基定位和底物结合的作用。与功能分配到活性位点的保守基团，我们讨论了缺乏活性的一些特征EAL结构域蛋白的分子基础和预测的磷酸二酯酶活性的细菌基因组中的大量的EAL结构域的催化机制的可能性。
EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO-1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg2+ ion. In addition to the five essential residues involved in Mg2+ binding, we propose that the essential residue E.. functions as a general base catalyst assisting the deprotonation of Mg2+ -coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in the k(cat) or K-m, leading us to propose their roles in residue positioning and substrate binding. With functions assigned to the conserved groups in the active site, we discuss the molecular basis for the lack of activity of some characterized EAL domain proteins and the possibility of predicting the phosphodiesterase activities for the vast number of EAL domains in bacterial genomes in light of the catalytic mechanism.