Abstract
Glutathionylspermidine synthetase (GspS) catalyzes glutathionylspermidine (Gsp) formation by coupling glutathione and spermidine with ATP hydrolysis. GspS belongs to the ATP-grasp superfamily. GspS from H. pylori (HpGspS) has been overexpressed and purified with a molecular weight of 45 kDa. HpGspS acts as a monomer in solution. The ATPase activity of HpGspS was determined as Km = 338 ± 21 μM and kcat = 9.2 ± 0.1 s-1. Three crystal structures of HpGspS, apo_HpGspS and HpGspS_ADP_Pi and HpGspS_AMPPNP complexes were determined at 2.4, 2.25 and 2.0 A resolution, respectively. The crystal structure of HpGspS was determined by multwavelenght anomalous dispersion (MAD). The overall structure of HpGspS shows a mixed α/β folding with an equilateral triangle shape, including an antiparallel β sheet, a parallel β sheet, and a lid domain. The ATP-binding site was located at central antiparallel β sheet and surrounded by four loops, 1oop β4-α3, loop β12-β13 (P-loop), 1oop β15-β16, and loop β18-β19. In HpGspS, residues Arg98, Asp100, Glu114, Asn116, Lys276, Lys308, Gly315 and Arg372 participating in the ATP binding, are all conserved among GspS. The adenine and ribose of ADP and AMPPNP are buried in a hydrophobic pocket which is formed by Leu113, Leu293, Ile306, Phe345, Leu348 and Ile377. The conformational changes of loop β12-β13 (P-loop), loop β18-β19, and loop β14-β15 (from lid domain) were observed in the presence of ADP_Pi and AMPPNP among apo and complexed HpGspS structures. The HpGspS_ADP_Pi complex structure might mimic the transition state for the hydrated phosphate and suggest how the γ-phosphate of ATP transfers to GSH. The ATP-binding (S1) site and putative glutathione (S2) as well as spermidine binding (S3) sites in HpGspS might illuminate the possible catalytic mechanism of HpGspS.