Discovery and Characterization of Pyridoxal 5′-Phosphate-Dependent Cycloleucine Synthases
Abner N. D. Abad, Kaushik Seshadri, Masao Ōhashi, David A. Delgadillo, Lygia S. de Moraes, Kyle K. Nagasawa, Mengting Liu, Samuel Johnson, Hosea M. Nelson, Yi Tang
Abstract
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.