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

Pyridoxal 5’-phosphate (PLP)-dependent enzymes are the most versatile biocatalysts for synthesizing non proteinogenic amino acids. a,a-disubstituted quaternary amino acids, such as 1-amino-1-cyclopentanecarboxylic 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 g-substitution. Whereas previously characterized g-replacement enzymes protonate the resulting a-carbon and release the acyclic amino acid, cycloleucine synthases can catalyze an additional, intramolecular aldol or Mannich reaction with the nucleophilic a-carbon to form the substituted cyclopentane. Overall, the [3+2]-annulation reaction can lead to 2-hydroxy or 2-aminocycloleucine products. These studies further expand the biocatalytic scope of PLP-dependent enzymes.