Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.
Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.
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DOI：
10.1021/acs.biochem.5b01227
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发表时间：
2016-02-16
期刊：
影响因子：
2.9
通讯作者：
Kurtz DM Jr
中科院分区：
文献类型：
--
作者：
Miner KD;Kurtz DM Jr
HD-GYP 构成金属依赖性 HD 磷酸水解酶超家族的一个亚类，催化环状二(3',5')-鸟苷单磷酸 (c-di-GMP) 转化为 5'-磷酸鸟苷基-(3'→5')-鸟苷 (pGpG) 和 GMP。迄今为止，唯一报道的也具有 c-di-GMP 磷酸二酯酶活性的 HD-GYP 晶体结构包含 His/羧酸盐连接的三铁活性位点。然而，其他结构和系统发育相关性表明一些 HD-GYP 含有二金属活性位点。在这里，我们提供的证据表明，来自海栖热袍菌的 HD-GYP c-di-GMP 磷酸二酯酶 TM0186 可以容纳二金属和三金属活性位点。我们表明，分离的含铁 TM0186 具有氧代/羧基桥联的二铁位点，并且还原（二价）形式对于催化 c-di-GMP 向 pGpG 的转化是必要且充分的，但 pGpG 向 GMP 的转化需要每个活性位点超过两种金属。用二价铁或锰获得了类似的c-di-GMP磷酸二酯酶活性。根据与几种假定的金属配体残基变体的活性相关性和分子动力学模拟，我们建议 TM0186 可以容纳二金属和三金属活性位点。我们的结果还表明，HD-GYP 子集中保守的 Glu 残基是三金属位点形成所必需的，并且也可以作为二金属位点的不稳定配体。考虑到 T. maritima 的厌氧生长要求，我们建议这种 HD-GYP 可以在体内与占据二金属和三金属位点的二价铁或锰一起发挥作用。
HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3′,5′)-guanosine monophosphate (c-di-GMP) to 5′-phosphoguanylyl-(3′→5′)-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.