Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme That Catalyzes C–C Bond Formation
Mengbin Chen, Chun‐Ting Liu, Yi Tang
Abstract
Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of l-amino acids at α, β, and γ positions. These enzymes are frequently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S,6S)-6-methyl pipecolate 1, from the biosynthetic pathway of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes the synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs γ-elimination of O-acetyl-l-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new Cγ–Cδ bond in 3 and complete the γ-substitution reaction. CndF displays promiscuity toward different β-keto carboxylate and esters. With use of an Aspergillus strain expressing CndF and CndE, feeding various alkyl-β-keto esters led to the biosynthesis of 6-substituted l-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.