Structural Basis for 3-Amino-3-carboxypropyl Transfer in Nocardicin Biosynthesis

S-Adenosyl-l-methionine (SAM) is well-known as a methyl donor for methyltransferases but also functions as a 3-amino-3-carboxypropyl (3-ACP) donor for 3-ACP transferases. NAT is a 3-ACP transferase which accepts β-lactam antibiotic nocardicin G (1) and SAM to produce isonocardicin C. Due to the lack of structural information about this enzyme, its reaction mechanism has not been fully identified. In this study, we report two X-ray crystal structures of NAT, including its apo and complex structure with 1 and SAH. Examination of them identified the structural basis for substrate recognition. Comprehensive approach integrating site-directed mutagenesis, thermal shift assay, MD simulation, and QM/MM calculation revealed that the Cα-amino group of SAM functions as a Brønsted base to enhance the nucleophilicity of the C6'-OH of 1, with the assistance of E143, thereby facilitating SN2 attack on the Cγ of SAM. This study presents structural and computational analysis leading to more precise understanding of 3-ACP transfer.