The C-terminal flexible region of branched-chain polyamine synthase facilitates substrate specificity and catalysis

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.