Interfaculty Institute of Biochemistry, University of Tübingen, Germany.
Interfaculty Institute for Microbiology and Infection Medicine, Department of Organismic Interactions, University of Tübingen, Germany.
FEBS J. 2016 Apr;283(7):1336-50. doi: 10.1111/febs.13673. Epub 2016 Feb 27.
To orchestrate a complex life style in changing environments, the filamentous cyanobacterium Nostoc punctiforme facilitates communication between neighboring cells through septal junction complexes. This is achieved by nanopores that perforate the peptidoglycan (PGN) layer and traverse the cell septa. The N-acetylmuramoyl-l-alanine amidase AmiC2 (Npun_F1846; EC 3.5.1.28) in N. punctiforme generates arrays of such nanopores in the septal PGN, in contrast to homologous amidases that mediate daughter cell separation after cell division in unicellular bacteria. Nanopore formation is therefore a novel property of AmiC homologs. Immunofluorescence shows that native AmiC2 localizes to the maturing septum. The high-resolution crystal structure (1.12 Å) of its catalytic domain (AmiC2-cat) differs significantly from known structures of cell splitting and PGN recycling amidases. A wide and shallow binding cavity allows easy access of the substrate to the active site, which harbors an essential zinc ion. AmiC2-cat exhibits strong hydrolytic activity in vitro. A single point mutation of a conserved glutamate near the zinc ion results in total loss of activity, whereas zinc removal leads to instability of AmiC2-cat. An inhibitory α-helix, as found in the Escherichia coli AmiC(E. coli) structure, is absent. Taken together, our data provide insight into the cell-biological, biochemical and structural properties of an unusual cell wall lytic enzyme that generates nanopores for cell-cell communication in multicellular cyanobacteria. The novel structural features of the catalytic domain and the unique biological function of AmiC2 hint at mechanisms of action and regulation that are distinct from other amidases.
The AmiC2-cat structure has been deposited in the Protein Data Bank under accession number 5EMI.
为了在不断变化的环境中协调复杂的生活方式，丝状蓝藻念珠藻通过隔膜连接复合体促进相邻细胞之间的通讯。这是通过在肽聚糖（PGN）层穿孔并穿过细胞隔膜的纳米孔实现的。在念珠藻中，N-乙酰胞壁酰-L-丙氨酸酰胺酶 AmiC2（Npun_F1846；EC 3.5.1.28）在隔膜 PGN 中生成这样的纳米孔阵列，与在单细胞细菌中介导细胞分裂后子细胞分离的同源酰胺酶形成对比。因此，纳米孔形成是 AmiC 同源物的新特性。免疫荧光显示，天然 AmiC2 定位于成熟的隔膜。其催化结构域（AmiC2-cat）的高分辨率晶体结构（1.12 Å）与已知的细胞分裂和 PGN 回收酰胺酶结构有很大的不同。一个宽阔而浅的结合腔允许底物轻松进入活性位点，其中含有一个必需的锌离子。AmiC2-cat 在体外表现出很强的水解活性。在靠近锌离子的保守谷氨酸上的单点突变导致完全丧失活性，而锌的去除导致 AmiC2-cat 的不稳定性。在大肠杆菌 AmiC（E. coli）结构中发现的抑制性α-螺旋不存在。总之，我们的数据提供了对一种不寻常的细胞壁裂解酶的细胞生物学、生化和结构特性的深入了解，该酶在多细胞蓝藻中生成纳米孔以进行细胞间通讯。催化结构域的新颖结构特征和 AmiC2 的独特生物学功能暗示了与其他酰胺酶不同的作用机制和调节机制。
AmiC2-cat 结构已在蛋白质数据银行中以 5EMI 的登录号进行了存储。
