Atomic coordinates and structure factors have been deposited in the Protein Data Bank with PDB codes 5XNF for apo-Tk-BpsA, 5XNH for the binary complex, and 5XNC for the ternary complex.

Branched‐chain polyamine synthase (BpsA) catalyzes sequential aminopropyl transfer from the donor, decarboxylated S‐adenosylmethionine (dcSAM), to the acceptor, linear‐chain polyamine, resulting in the production of a quaternary‐branched polyamine via tertiary branched polyamine intermediates. Here, we analyzed the catalytic properties and X‐ray crystal structure of Tth‐BpsA from Thermus thermophilus and compared them with those of Tk‐BpsA from Thermococcus kodakarensis, which revealed differences in acceptor substrate specificity and C‐terminal structure between these two enzymes. To investigate the role of the C‐terminal flexible region in acceptor recognition, a region (QDEEATTY) in Tth‐BpsA was replaced with that in Tk‐BpsA (YDDEESSTT) to create chimeric Tth‐BpsA C9, which showed a severe reduction in catalytic efficiency toward N4‐aminopropylnorspermidine, but not toward N4‐aminopropylspermidine, mimicking Tk‐BpsA substrate specificity. Tth‐BpsA C9 Tyr346 and Thr354 contributed to discrimination between tertiary branched‐chain polyamine substrates, suggesting that the C‐terminal region of BpsA recognizes acceptor substrates. Liquid chromatography‐tandem mass spectrometry analysis on a Tk‐BpsA reaction mixture with dcSAM revealed two aminopropyl groups bound to two of five aspartate/glutamate residues (Glu339, Asp342, Asp343, Glu344, and Glu345) in the C‐terminal flexible region. Mutating each of these five amino acid residues to asparagine/glutamine resulted in a slight decrease in activity. The quadruple mutant D342N/D343N/E344Q/E345Q exhibited a severe reduction in catalytic efficiency, suggesting that these aspartate/glutamate residues function to receive aminopropyl chains. In addition, the X‐ray crystal structure of the Tk‐BpsA ternary complex bound to N4‐bis(aminopropyl)spermidine revealed that Asp126 and Glu259 interacted with the aminopropyl moiety in N4‐aminopropylspermidine.