Putrescine-bound structure and multi-temperature crystallography provide new insights into ligand binding and conformational heterogeneity in spermidine synthase.

Spermidine synthase (SPDS) is an important enzyme in the pathway for the biosynthesis of polyamines, and it synthesizes the polyamine spermidine from putrescine (PUT) and decarboxylated S-adenosylmethionine. This pathway is a promising target for antiproliferative therapies because of the functions of polyamines in cellular proliferation and differentiation. Higher polyamine levels are linked to cancer and cell proliferation. Previous structures of the enzyme were solved at cryogenic temperatures. Here, we report a new SPDS structure in complex with PUT (SPDS-PUT) and multitemperature crystallography of SPDS complexed with PUT and 5-deoxy-5-methylthioadenosine (MTA) at 100, 273, and 293 K to study its structure and dynamics. The results revealed that the occupancy of the active site is important for the extent of stabilization in the gatekeeping loop. The SPDS-PUT structure revealed putrescine binding in the absence of MTA, providing insight into its interactions within the active site and the conformational state of the gatekeeping loop. The binding of PUT alone was not enough to stabilize the gatekeeping loop, and greater stabilization of the loop occurred in the presence of MTA alone compared to PUT alone. The observed structural asymmetry between the gatekeeping loops of SPDS reflects differences in ligand binding between chains. These findings expand our understanding of the conformational landscape of SPDS.