Cao Hai-Yan, Wang Peng, Xu Fei, Li Ping-Yi, Xie Bin-Bin, Qin Qi-Long, Zhang Yu-Zhong, Li Chun-Yang, Chen Xiu-Lan
State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan, China.
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Front Microbiol. 2017 Oct 17;8:2034. doi: 10.3389/fmicb.2017.02034. eCollection 2017.
Microbial cleavage of dimethylsulfoniopropionate (DMSP) producing dimethyl sulfide (DMS) and acrylate is an important step in global sulfur cycling. Acrylate is toxic for cells, and thus should be metabolized effectively for detoxification. There are two proposed pathways for acrylate metabolism in DMSP-catabolizing bacteria, the AcuN-AcuK pathway and the PrpE-AcuI pathway. AcuH is an acryloyl-CoA hydratase in DMSP-catabolizing bacteria and can catalyze the hydration of toxic acryloyl-CoA to produce 3-hydroxypropionyl-CoA (3-HP-CoA) in both the AcuN-AcuK pathway and the side path of the PrpE-AcuI pathway. However, the structure and catalytic mechanism of AcuH remain unknown. Here, we cloned a putative gene from ISM, a typical DMSP-catabolizing bacterium, and expressed it (AcuH) in . The activity of AcuH toward acryloyl-CoA was detected by liquid chromatography-mass spectrometry (LC-MS), which suggests that AcuH is a functional acryloyl-CoA hydratase. Then we solved the crystal structure of AcuH. Each asymmetric unit in the crystal of AcuH contains a dimer of trimers and each AcuH monomer contains an N-terminal domain (NTD) and a C-terminal domain (CTD). There are three active centers in each trimer and each active center is located between the NTD of a subunit and the CTD of the neighboring subunit. Site-directed mutagenesis analysis indicates that two highly conserved glutamates, Glu112 and Glu132, in the active center are essential for catalysis. Based on our results and previous research, we analyzed the catalytic mechanism of AcuH to hydrate acryloyl-CoA, in which Glu132 acts as the catalytic base. This study sheds light on the mechanism of acrylate detoxification in DMSP-catabolizing bacteria.
二甲基巯基丙酸内盐（DMSP）经微生物裂解生成二甲基硫醚（DMS）和丙烯酸酯是全球硫循环中的重要一步。丙烯酸酯对细胞有毒，因此需要有效代谢以解毒。在利用DMSP的细菌中，目前提出了两条丙烯酸酯代谢途径，即AcuN - AcuK途径和PrpE - AcuI途径。AcuH是利用DMSP的细菌中的一种丙烯酰辅酶A水合酶，在AcuN - AcuK途径和PrpE - AcuI途径的侧支中，它都能催化有毒的丙烯酰辅酶A水合生成3 - 羟基丙酰辅酶A（3 - HP - CoA）。然而，AcuH的结构和催化机制尚不清楚。在此，我们从典型的利用DMSP的细菌ISM中克隆了一个假定基因，并在大肠杆菌中表达了该基因（AcuH）。通过液相色谱 - 质谱联用（LC - MS）检测了AcuH对丙烯酰辅酶A的活性，这表明AcuH是一种有功能的丙烯酰辅酶A水合酶。然后我们解析了AcuH的晶体结构。AcuH晶体中的每个不对称单元包含一个三聚体二聚体，每个AcuH单体包含一个N端结构域（NTD）和一个C端结构域（CTD）。每个三聚体中有三个活性中心，每个活性中心位于一个亚基的NTD和相邻亚基的CTD之间。定点突变分析表明，活性中心的两个高度保守的谷氨酸残基Glu112和Glu132对催化至关重要。基于我们的研究结果和之前的研究，我们分析了AcuH催化丙烯酰辅酶A水合的机制，其中Glu132作为催化碱。这项研究揭示了利用DMSP的细菌中丙烯酸酯解毒的机制。
