Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog
Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog
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DOI：
10.1073/pnas.1019480108
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发表时间：
2011-03-08
影响因子：
11.1
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
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作者：
Bonnefond, Luc;Arai, Taiga;Nureki, Osamu
环二肽是由许多细菌生物合成的次级代谢产物，具有广泛的生物活性。最近，一类新的小蛋白质，命名为环二肽合成酶（CDPS），这是不相关的典型的非核糖体肽合成酶，被证明可以产生几个环二肽，使用氨酰-tRNA作为底物。发现结核分枝杆菌CDPS Rv2275通过形成氨酰-酶中间体产生环二酪氨酸，并且具有与Ic类氨酰-tRNA合成酶（aaRS）相似的结构和寡聚状态。然而，差的序列保守性CDPS之间提出了问题的架构和催化机制的确定同源日志。在这里，我们报告地衣芽孢杆菌CDPS YvmC-Blic的晶体结构，在载脂蛋白的形式和复杂的底物模拟物，在1.7 - 2.4埃的分辨率。YvmC-Blic结构也表现出与Ic类aaRS催化结构域的相似性。我们的突变分析证实了一组残基之间的保守残基定位在催化口袋中的环二亮氨酸形成的重要性。我们的生物化学数据表明，YvmC-Blic结合tRNA并产生作为单体的环二亮氨酸。我们还能够检测到氨酰-酶反应中间体的存在，但不是二肽tRNA中间体，其存在被假定为Rv2275。相反，我们的研究结果支持YvmC-Blic的顺序催化机制，通过保守的丝氨酸残基在酶上连续连接两个亮氨酸残基。总之，我们的研究结果表明，所有的CDPS酶共享一个共同的aaRS样的架构和催化机制，涉及形成的酶结合的中间体。
Cyclodipeptides are secondary metabolites biosynthesized by many bacteria and exhibit a wide array of biological activities. Recently, a new class of small proteins, named cyclodipeptide synthases (CDPS), which are unrelated to the typical nonribosomal peptide synthetases, was shown to generate several cyclodipeptides, using aminoacyl-tRNAs as substrates. The Mycobacterium tuberculosis CDPS, Rv2275, was found to generate cyclodityrosine through the formation of an aminoacyl-enzyme intermediate and to have a structure and oligomeric state similar to those of the class Ic aminoacyl-tRNA synthetases (aaRSs). However, the poor sequence conservation among CDPSs has raised questions about the architecture and catalytic mechanism of the identified homo-logs. Here we report the crystal structures of Bacillus licheniformis CDPS YvmC-Blic, in the apo form and complexed with substrate mimics, at 1.7-2.4-angstrom resolutions. The YvmC-Blic structure also exhibits similarity to the class Ic aaRSs catalytic domain. Our mutational analysis confirmed the importance of a set of residues for cyclodileucine formation among the conserved residues localized in the catalytic pocket. Our biochemical data indicated that YvmC-Blic binds tRNA and generates cyclodileucine as a monomer. We were also able to detect the presence of an aminoacyl-enzyme reaction intermediate, but not a dipeptide tRNA intermediate, whose existence was postulated for Rv2275. Instead, our results support a sequential catalytic mechanism for YvmC-Blic, with the successive attachment of two leucine residues on the enzyme via a conserved serine residue. Altogether, our findings suggest that all CDPS enzymes share a common aaRS-like architecture and a catalytic mechanism involving the formation of an enzyme-bound intermediate.