麩胺基硫亞精胺合成酶 (GspS) 藉由三磷酸腺苷 (ATP) 的水解來結合榖胱甘肽 (GSH) 與亞精胺 (spermidine) 形成麩胺基硫亞精胺 (Gsp)，GspS是屬於ATP結合家族的成員。本實驗中幽門螺旋桿菌的麩胺基硫亞精胺合成酶 (HpGspS) 被大量表現以及純化，其分子量為45 kDa，並且在水溶液中是以單體存在。經由酵素活性分析結果顯示HpGspS的ATP水解活性為Km = 338 ± 21 μM 及kcat = 9.2 ± 0.1 s-1。HpGspS晶體 (apo_HpGspS)，HpGspS與二磷酸腺苷、磷酸的複合物晶體 (HpGspS_ADP_Pi) 及HpGspS與亞胺三磷酸腺苷的複合物晶體 (HpGspS_AMPPNP) 的解析度分別為2.4、2.25 及2.0埃。HpGspS的晶體結構是以硒 (Selenium) 的多波長異常散射法解析得到，其三度空間結構為α螺旋/β摺疊混合形成外觀像是正三角形的形狀，包含蓋子區域 (lid domain)、反向平行β折疊 (antiparallel β sheet) 與同向平行β折疊 (parallel β sheet)。ATP的結合位置位於中心反向平行β折疊之上，由四個環狀區域 (1oop β4-α3, loop β12-β13 (P-loop), 1oop β15-β16, and loop β18-β19) 所環繞組成。參與ADP_Pi及AMPPNP結合的胺基酸Arg98、Asp100、Glu114、Asn116、Lys276、Lys308、Gly315、Arg372在GspS之間都具有高度的保留性。另外，ADP及AMPPNP的腺嘌呤 (adenine) 與五碳糖 (ribose) 被疏水性的胺基酸Leu113, Leu293, Ile306, Phe345, Leu348 and Ile377所埋藏。從apo_HpGspS、HpGspS_ADP_Pi及HpGspS_AMPPNP的三度空間結構觀察到三個環狀區域 (loop β12-β13 (P-loop), loop β18-β19, and loop β14-β15) 的構型變化。在HpGspS_ADP_Pi的複合物晶體結構中，其被水解的磷酸或許能模仿HpGspS催化時的過渡狀態，推測ATP上的磷酸是如何轉移到GSH上。此外，從ATP的結合位置和假定的GSH及spermidine的結合位置，或許可以闡明HpGspS催化合成反應可能的作用機制。

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.

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