Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic β2 and β7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.

中文翻译：

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环二肽合酶与氨酰化 tRNA 底物相互作用的结构基础

环二肽合酶 (CDPS) 通过以顺序机制使用两种氨酰-tRNA (aa-tRNA) 底物来催化各种环二肽的合成。在这里，我们通过凝胶过滤和电泳迁移率变化测定研究了苯丙氨酰-tRNAPhe 与来自巨大球孢菌 (Cglo-CDPS) 的 CDPS 的结合。我们将 Cglo-CDPS:Phe-tRNAPhe 复合物的晶体结构确定为 5 Å 分辨率，并使用小角 X 射线散射 (SAXS) 在溶液中进一步研究它。数据显示，aa-tRNA 受体茎的大沟通过属于 XYP 亚家族的 CDPS 的基本 β2 和 β7 链与酶相互作用。CCA 末端的弯曲使氨基酸部分能够位于 P1 口袋中，而末端 A76 腺苷占据 P2 口袋。这种定位表明本结构说明了第一个 aa-tRNA 的结合。在细胞中，CDPS 和延伸因子 EF-Tu 共享氨酰化 tRNA 作为底物。本研究表明 CDPSs 和 EF-Tu 与 tRNA 的相对侧相互作用。这可以解释 CDPS 如何从经典核糖体蛋白质合成中劫持 aa-tRNA。