Kaitocephalin (KCP) is a fungal neuroactive natural product bearing a peptide-like yet nonpeptidic amino acid-derived scaffold in which amino acid-like units are connected by C-C bonds rather than peptide bonds. The enzymatic construction of this unusual scaffold has remained unresolved. Here, we identify KpbH as a PLP-dependent enzyme that couples pyrroline-5-carboxylate, generated from L-ornithine, with L-aspartate to form (2S,5R)-5-((S)-2-amino-2-carboxyethyl)pyrrolidine-2-carboxylic acid (ACPCA), which corresponds to the nonpeptidic Ala-Pro substructure of KCP. D2O-labeling experiments showed enzyme-controlled, solvent-derived deuterium incorporation at C7 of ACPCA, supporting a decarboxylative Mannich-type mechanism. Feeding of a deuterium-enriched ACPCA-containing reaction mixture to the KCP-producing fungus Eupenicillium shearii resulted in deuterium incorporation into KCP, linking ACPCA to KCP biosynthesis. These results identify KpbH as the first native PLP-dependent enzyme that catalyzes an L-aspartate-dependent decarboxylative Mannich-type C-C bond-forming reaction and reveal a biosynthetic strategy for constructing a noncanonical amino acid-like C-C bond scaffold.
1. Kaitocephalin (KCP) 是一种真菌神经活性天然产物，其具有肽样但非肽的氨基酸衍生骨架，其中类氨基酸单元通过 C-C 键而非肽键连接。这种不寻常骨架的酶促构建机制一直未被阐明。在此，我们鉴定出 KpbH 是一种 PLP 依赖性酶，它将 L-鸟氨酸生成的吡咯啉-5-羧酸与 L-天冬氨酸偶联，形成 (2S,5R)-5-((S)-2-氨基-2-羧乙基)吡咯烷-2-羧酸 (ACPCA)，该产物对应于 KCP 的非肽 Ala-Pro 亚结构。D2O 标记实验显示，酶控制的溶剂衍生氘掺入 ACPCA 的 C7 位，支持了脱羧曼尼希型机制。将富含氘的含 ACPCA 反应混合物喂给产 KCP 的真菌 Eupenicillium shearii，导致氘掺入 KCP 中，将 ACPCA 与 KCP 生物合成联系起来。这些结果确定 KpbH 是首个天然 PLP 依赖性酶，催化 L-天冬氨酸依赖的脱羧曼尼希型 C-C 键形成反应，并揭示了构建非典型类氨基酸 C-C 键骨架的生物合成策略。
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Noguchi, T. et al. · CC-BY 4.0