Capreomycin (CMN) and viomycin (V10) are non-ribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including L-2,3-diaminopropionic acid (L-Dap), beta-ureidodehydroalanine, L-capreomycidine, and beta-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of L-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-L-Ser (OPS) and L-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD(+)-dependent oxidative hydrolysis by CmnK to generate L-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD(+)-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate alpha-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce L-Dap. These findings modify the original proposed pathway and provide insights into L-Dap formation in the biosynthesis of other related natural products.