Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States.
Biochemistry. 2022 Sep 6;61(17):1801-1809. doi: 10.1021/acs.biochem.2c00269. Epub 2022 Jul 28.
Cyclic dinucleotides (CDNs) are signaling molecules involved in the immune response and virulence factor production. CDN cellular levels are fine-tuned by metal-dependent phosphodiesterases (PDEs), among which HD-GYPs make up a subclass of the larger HD-domain protein superfamily. The human pathogen () encodes nine HD-GYPs, one of which is V-cGAP3 (or VCA0931). V-cGAP3 acts on c-di-GMP and 3'3'c-GAMP, and this activity is related to bacterial infectivity. However, the extant chemical makeup of the V-cGAP3 cofactor and steady state parameters have not been established. Employing electron paramagnetic resonance and Mössbauer spectroscopy in tandem with elemental analyses and activity assays, we demonstrate that V-cGAP3 coordinates different dimetal cofactors with variable activities. Mn and Fe afford c-di-GMP hydrolysis with the highest observed rates, while c-GAMP hydrolysis is selectively dependent on Mn. V-cGAP3 has a single functional domain, and this simple architecture allows us to examine the roles of characteristic conserved residues in catalysis. Substitution of the adjacent to the active site GYP residue triad and the specifically conserved in HD-domain PDEs fifth histidine ligand (i.e., H371 in V-cGAP3) with alanines severely compromises CDN hydrolysis but only modestly affects cofactor incorporation. Our data are consistent with V-cGAP3 being the major regulator of 3'3'c-GAMP hydrolysis in and delineate the importance of specific residues in tuning activity in HD-GYPs in general. We propose that HD-GYPs exhibit diversity in their metallocofactors and substrates, which may serve to increase their functional potential in regulatory pathways or allow for PDE activity upon adaptation of the parent organism to diverse environmental niches.
环二核苷酸 (CDNs) 是参与免疫反应和毒力因子产生的信号分子。CDN 细胞水平受金属依赖性磷酸二酯酶 (PDE) 精细调节，其中 HD-GYPs 构成较大的 HD 结构域蛋白超家族的一个亚类。人类病原体 () 编码九个 HD-GYPs，其中一个是 V-cGAP3（或 VCA0931）。V-cGAP3 作用于 c-di-GMP 和 3'3'c-GAMP，其活性与细菌感染力有关。然而，V-cGAP3 辅因子的现有化学组成和稳态参数尚未确定。我们采用电子顺磁共振和穆斯堡尔光谱学与元素分析和活性测定相结合的方法，证明 V-cGAP3 与不同的二价金属辅因子协调，具有不同的活性。Mn 和 Fe 提供了最高观察到的 c-di-GMP 水解速率，而 c-GAMP 水解则选择性地依赖于 Mn。V-cGAP3 具有单个功能域，这种简单的结构使我们能够研究特征保守残基在催化中的作用。用丙氨酸替代活性位点 GYP 残基三联体和在 HD 结构域 PDEs 中特异性保守的第五个组氨酸配体（即 V-cGAP3 中的 H371），严重损害了 CDN 水解，但仅适度影响辅因子的结合。我们的数据与 V-cGAP3 是 中 3'3'c-GAMP 水解的主要调节剂一致，并阐明了特定残基在调节 HD-GYPs 活性中的重要性。我们提出，HD-GYPs 在其金属辅因子和底物中表现出多样性，这可能增加它们在调节途径中的功能潜力，或者允许 PDE 活性在母体生物适应不同的环境小生境时发挥作用。
