Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metal-dependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3′3′c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized Fe^IIFe^III state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent k_cat and K_M (but not k_cat/K_M), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.

中文翻译：

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HD- [HD-GYP]磷酸二酯酶：HD-GYP家族中的活性和进化多样化。

环状二核苷酸是调节许多过程的信号分子，包括免疫应答和毒力因子的产生。它们在细菌中的细胞水平可通过金属依赖性磷酸二酯酶（即EAL和HD-GYP蛋白）进行微调，其中HD-GYP属于较大的HD域超家族。在这项研究中，我们首先关注由两个HD域组成的HD- [HD-GYP]亚家族的催化性能以及金属离子和底物的范围。我们将SO3491确定为VCA0681的同源物，并且是HD- [HD-GYP]的第二个示例。两种蛋白质都水解c-di-GMP和3'3'c-GAMP并协调各种金属离子，但只有Fe和少量的Co支持水解。蛋白质仅以^二铁形式有活性，而不是单电子氧化的Fe ^II Fe。^III状态。尽管C端HD-GYP域对于活性至关重要，但N端HD域的作用仍然未知。我们显示N末端位点对于蛋白质稳定性很重要，会影响单个表观k _cat和K _M（但不影响k _cat / K _M），并且无法结合c-di-GMP，因此排除了c-di-GMP参与环二核苷酸感测。我们着手进行系统发育分析，以检查HD- [HD-GYP]与其余HD-GYP的分布和功能关系。系统发育提供了一个关联图，该图绘制了HD-GYP的进化和功能多样化之间的联系，用作预测金属因子的化学性质，活性水平和反应结果的模板。