Structure and Mechanism of the Pyridoxal 5′-Phosphate-Dependent Amino Acid γ-Substitution Enzyme in 6-Alkyl-Pipecolate Biosynthesis

The pyridoxal 5′-phosphate (PLP)-dependent enzyme CndF catalyzes the C–C bond formation at the γ-position of amino acid to afford 6-alkyl-pipecolates. With substrate promiscuity and stereocontrol, CndF has shown potential in synthetic chemistry. However, lacking a detailed mechanistic understanding, the exploitation of such potential is hindered. Herein, we report the complex structure of the tridomain enzyme CndF in complex with PLP, l-homoserine (HSE), and ethyl acetoacetate (EAA), where EAA binds to R529 through hydrogen bonding while HSE forms geminal diamine with PLP and residue K366. Mutagenesis, computational studies, and spectroscopic data support an elimination-addition type mechanism for γ-substitution happening on the ketimine intermediate, in which K366 serves as a general acid–base catalyst. Our studies will pave the road for broadening the substrate scope of CndF and expanding the types of enzymatic C–C bond formation at the γ-position of amino acids.