Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase.
Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase.
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GdpP 催化域的结构和生化表征揭示了 DHH/DHHA1 磷酸二酯酶的统一水解机制。
DOI：
10.1042/bcj20170739
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发表时间：
2018
影响因子：
4.1
通讯作者：
Lichuan Gu
中科院分区：
文献类型：
--
作者：
Fneg Wang;Qing He;Kaixuan Su;Ti;i Wei;Sujuan Xu;Lichuan Gu
Asp-His-His和Asp-His-His-associated（DHH/DHHA 1）结构域磷酸二酯酶（PDE）催化环二磷酸腺苷（c-di-AMP）的降解，根据最终产物[5 '-磷酸腺苷酰腺苷（5'-pApA）或AMP]，可分为两个亚家族。在先前的研究中，我们揭示了Rv 2837 c，一种独立的DHH/DHHA 1 PDE，采用5 '-pApA内部翻转机制来产生AMP。然而，为什么膜结合DHH/DHHA 1 PDE只能将c-di-AMP降解为5 '-pApA仍然不清楚。在这里，我们报告了GdpP（GdpP-C）的DHH/DHHA 1结构域的晶体结构，以及与c-di-AMP、环二鸟苷一磷酸（c-di-GMP）和5 '-pApA复合的结构。结构分析表明，GdpP-C结合核苷酸底物的方式与Rv 2837 c在底物结合位置方面完全不同。因此，DHH/DHHA 1 PDE的核苷酸结合位点被组织成三个（C、G和R）亚位点。对于GdpP-C，在C和G位点，c-di-AMP结合并降解为5 '-pApA，其G位点决定核苷酸特异性。为了进一步降解为AMP，5 '-pApA必须滑入C和R位点以进行翻转和水解，如在Rv 2837 c中。随后的诱变和酶的研究GdpP-C和Rv 2837 c揭示了完整的翻转过程，并揭示了一个统一的催化机制的DHH/DHHA 1 PDE亚家族的成员。
The Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterases (PDEs) that catalyze degradation of cyclic di-adenosine monophosphate (c-di-AMP) could be subdivided into two subfamilies based on the final product [5'-phosphadenylyl-adenosine (5'-pApA) or AMP]. In a previous study, we revealed that Rv2837c, a stand-alone DHH/DHHA1 PDE, employs a 5'-pApA internal flipping mechanism to produce AMPs. However, why the membrane-bound DHH/DHHA1 PDE can only degrade c-di-AMP to 5'-pApA remains obscure. Here, we report the crystal structure of the DHH/DHHA1 domain of GdpP (GdpP-C), and structures in complex with c-di-AMP, cyclic di-guanosine monophosphate (c-di-GMP), and 5'-pApA. Structural analysis reveals that GdpP-C binds nucleotide substrates quite differently from how Rv2837c does in terms of substrate-binding position. Accordingly, the nucleotide-binding site of the DHH/DHHA1 PDEs is organized into three (C, G, and R) subsites. For GdpP-C, in the C and G sites c-di-AMP binds and degrades into 5'-pApA, and its G site determines nucleotide specificity. To further degrade into AMPs, 5'-pApA must slide into the C and R sites for flipping and hydrolysis as in Rv2837c. Subsequent mutagenesis and enzymatic studies of GdpP-C and Rv2837c uncover the complete flipping process and reveal a unified catalytic mechanism for members of both DHH/DHHA1 PDE subfamilies.