In the biosynthesis of the lincosamide antibiotic celesticetin, the condensation enzyme CcbD generates the lincosamide pharmacophore by forming an amide bond between the carrier protein (CP)-tethered proline and ergothioneine-conjugated thiooctose. Although the function of CcbD has been investigated, its substrate specificity, structures and catalytic mechanisms remain unclear. Here we show the structure–function analyses of CcbD. Our biochemical analysis revealed that CcbD exhibits promiscuous substrate specificity towards CP-tethered acyl substrates to generate unnatural lincosamides. Furthermore, structural analyses indicated that CcbD possesses an unusual overall fold, while the N-terminal region shows weak similarity to cysteine proteases. Thus, CcbD, like cysteine proteases, utilizes the Cys-His-Glu catalytic triad to form amide bonds in a CP-dependent manner, which is significantly different from other known amide bond-forming enzymes. Furthermore, the structures of the CcbD/thiooctose complex and the cross-linked CcbD/CcbZ-CP complex, as well as structure-based mutagenesis, revealed the intimate structural details of the CP-dependent amide bond formation reaction.

在林可酰胺类抗生素 celesticetin 的生物合成中，缩合酶 CcbD 通过在载体蛋白 (CP) 连接的脯氨酸和麦角硫因偶联的硫辛糖之间形成酰胺键生成林可酰胺药效团。尽管已经研究了 CcbD 的功能，但其底物特异性、结构和催化机制仍不清楚。在这里，我们展示了 CcbD 的结构-功能分析。我们的生化分析表明，CcbD 对 CP 束缚的酰基底物表现出混杂的底物特异性，以产生非天然的林可酰胺。此外，结构分析表明 CcbD 具有不寻常的整体折叠，而 N 末端区域与半胱氨酸蛋白酶的相似性较弱。因此，CcbD 与半胱氨酸蛋白酶一样，利用 Cys-His-Glu 催化三联体以 CP 依赖性方式形成酰胺键，这与其他已知的酰胺键形成酶显着不同。此外，CcbD/硫辛糖复合物和交联的 CcbD/CcbZ-CP 复合物的结构，以及基于结构的诱变，揭示了 CP 依赖性酰胺键形成反应的密切结构细节。