A Pyridoxal 5′-Phosphate-Dependent Decarboxylative Mannich Reaction Initiates Construction of the Nonpeptidic Scaffold of Kaitocephalin

Abstract 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 pyridoxal 5′-phosphate (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.