Discovery and Characterization of Pyridoxal 5′-Phosphate-Dependent Cycloleucine Synthases

Pyridoxal 5′-phosphate (PLP)-dependent enzymes are the most versatile biocatalysts for synthesizing nonproteinogenic amino acids. α,α-Disubstituted quaternary amino acids, such as 1-aminocyclopentane-1-carboxylic acid (cycloleucine), are useful building blocks for pharmaceuticals. In this study, starting with the biosynthesis of fusarilin A, we discovered a family of PLP-dependent enzymes that can facilitate tandem carbon–carbon forming steps to catalyze an overall [3 + 2]-annulation. In the first step, the cycloleucine synthases use SAM as the latent electrophile and an in situ-generated enamine as the nucleophile for γ-substitution. Whereas previously characterized γ-replacement enzymes protonate the resulting α-carbon and release the acyclic amino acid, cycloleucine synthases can catalyze an additional, intramolecular aldol or Mannich reaction with the nucleophilic α-carbon to form the substituted cyclopentane. Overall, the net [3 + 2]-annulation reaction can lead to 2-hydroxy or 2-aminocycloleucine products. These studies further expand the biocatalytic scope of PLP-dependent enzymes.