Crystal structure of the nosiheptide-resistance methyltransferase of Streptomyces actuosus.
Crystal structure of the nosiheptide-resistance methyltransferase of Streptomyces actuosus.
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DOI：
10.1021/bi1005915
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发表时间：
2010-07
期刊：
影响因子：
2.9
通讯作者：
Huirong Yang;Zhe Wang;Yan Shen;Ping Wang;Xu Jia;Liang Zhao;P. Zhou;R. Gong;Ze Li;Ying Yang;Dongrong Chen;A. Murchie;Yanhui Xu
中科院分区：
文献类型：
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作者：
Huirong Yang;Zhe Wang;Yan Shen;Ping Wang;Xu Jia;Liang Zhao;P. Zhou;R. Gong;Ze Li;Ying Yang;Dongrong Chen;A. Murchie;Yanhui Xu
链霉菌（Streptomyces actusus）的Nosiheptide-resistance methyltransferase （NHR）是SpoU家族的IV类甲基转移酶，在大肠杆菌中甲基化23S rRNA的核苷酸腺苷，对应于A1067。这种甲基化对于抵抗诺西肽是必不可少的，诺西肽是一种硫肽抗生素，由诺西肽产生菌株S. actuosus产生。本文报道了NHR和NHR在2.0和2.1 A分辨率下与SAM （s -腺苷-l-蛋氨酸）配合物的晶体结构。NHR形成功能性同二聚体，甲基转移酶活性需要二聚体。NHR单体由n端RNA结合域（NTD）和c端催化域（CTD）组成。总的来说，NHR的结构表明，甲基转移酶的活性是通过NTD“读取”RNA底物并使用CTD“添加”甲基来实现的。综合诱变和甲基转移酶活性分析揭示了CTD中SAM结合的关键区域和NTD中RNA识别所必需的环（L1和L3）。最后，在结构分析和生化分析的基础上，提出了NHR识别23S rRNA的催化机理和结构模型。因此，我们的系统结构研究揭示了诺西肽抗性甲基转移酶对底物的识别和修饰。
Nosiheptide-resistance methyltransferase (NHR) of Streptomyces actuosus is a class IV methyltransferase of the SpoU family and methylates 23S rRNA at nucleotide adenosine corresponding to A1067 in Escherichia coli. Such methylation is essential for resistance against nosiheptide, a sulfur peptide antibiotic, which is produced by the nosiheptide-producing strain, S. actuosus. Here, we report the crystal structures of NHR and NHR in complex with SAM (S-adenosyl-l-methionine) at 2.0 and 2.1 A resolution, respectively. NHR forms a functional homodimer, and dimerization is required for methyltransferase activity. The monomeric NHR is comprised of the N-terminal RNA binding domain (NTD) and the C-terminal catalytic domain (CTD). Overall, the structure of NHR suggests that the methyltransferase activity is achieved by "reading" the RNA substrate with NTD and "adding" methyl group using CTD. Comprehensive mutagenesis and methyltransferase activity assays reveal critical regions for SAM binding in CTD and loops (L1 and L3) essential for RNA recognition in NTD. Finally, the catalytic mechanism and structural model that NHR recognizes 23S rRNA is proposed based on the structural and biochemical analyses. Thus, our systematic structural studies reveal the substrate recognition and modification by the nosiheptide-resistance methyltransferase.