幽門螺旋桿菌亞精胺合成酶與受質同源衍生物及抑制劑複合物晶體結構之研究

Crystal Structure Studies of Helicobacter pylori Spermidine Synthase in Complex with the Substrate Analogue and Potent Inhibitor

林建志(Chien-Chih Lin)

指導教授 ： 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2008年)

摘要

亞精胺合成□將脫羧腺甘甲硫胺酸的丙胺基轉移到腐胺上，進而形成亞精胺與去氧甲硫腺甘酸。在之前，我們實驗室已經成功利用X光晶體繞射技術多波長異常色散法解出含重金屬硒的甲硫胺酸衍生物幽門螺旋桿菌亞精胺合成□晶體結構。幽門螺旋桿菌亞精胺合成□由兩個功能區組成，分別為N端的摺版區和C端的Rossmann-like 折疊功能區。而受質結合部位則位於N與C端功能區之間的一個活化部位。與其它亞精胺合成□結構比較，幽門螺旋桿菌亞精胺合成□的活化部位具有較大的空間，較低的酸性，以及許多非保留性的胺基酸。我們利用類似受質的腺甘甲硫胺酸和有抑制劑功能的去氧甲硫腺甘酸跟幽門螺旋桿菌亞精胺合成□作複合物的形成。這兩種不同的複合物晶體HpPAPT-SAM 、HpPAPT-MTA分別可以達到2.1 和2.0 □ 的解析度。而這兩個不同之受質/產物的結合位置與方位都非常的類似。但是這個結合部位與我們之前發表的期刊中所假設的活化部位並大不一致，其中期刊裡假設的結合區包含三個部份，分別有與腺甘結合的胺基酸(N29, Q101, A102, H128, L146, Q147, E148, and P149)，與丙胺基結合的胺基酸(E58, S59, D83)，最後是與腐胺結合的胺基酸(M44, L49, D80, G81, S206, and I211)1。而新飄移之結合部位是更接近Rossmann-like 折疊功能區，也就是坐落於丙胺基和腐胺這兩個結合區，而其中只有E58和D83符合在期刊中提到的活化部位裡頭。另外有其他的胺基酸例如F51, F54, R203以及L205圍繞在腺甘甲硫胺酸與去氧甲硫腺甘酸週遭。除此之外，因為亞精胺合成□對細胞生存是必需的所以藉由結構上的資訊，幽門螺旋桿菌亞精胺合成□可以作為藥物設計的目標。

關鍵字

亞精胺合成□

並列摘要

Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from S-adenosylmethionine (dcSAM) to putrescine, leading to form spermindine and 5’-deoxy-5’-methylthioadenosine (MTA). The structure of Helicobacter pylori spermidine synthase (HpPAPT) was solved by our lab previously, using multiwavelengh anomalous dispersion (MAD) from selenomethionine (SeMet)-containing crystals. HpPAPT consisted of two domains, an N-terminal β-strand domain including 6β-strands, and a C-terminal Rossmann-like domain. Moreover, the substrate binding site might locate in a deep binding pocket between the N- and C- terminal domains. Structural comparison with other PAPTs showed that HpPAPT has a unique binding pocket between two domains, numerous non-conserved residues, a less acidic electrostatic surface potential, and a large buried space within the structure. We used the substrate analogue S-adenosylmethionine (SAM) and the potent inhibitor 5’-deoxy-5’-methylthioadenosine (MTA) in complex with HpPAPT. Two complex crystals, HpPAPT-SAM and HpPAPT-MTA, were determined at 2.1 and 2.0 □, respectively. The binding site and orientation of the two bound substrate/product of the HpPAPT are very similar. But there is difference between the shifted binding site and the putative active site, which included three parts, adenosyl moiety binding residues (N29, Q101, A102, H128, L146, Q147, E148, and P149), aminopropyl group binding residues (E58, S59, D83) and putrescine binding residues (M44, L49, D80, G81, S206, and I211)1. This shifted binding pocket is located in aminopropyl group and putrescine binding part, which is close to Rossmann-like domain, and only two residues, E58 and D83 are corresponding to the putative active site. There are other residues, F51, F54, R203 and L205 involved in SAM or MTA binding site. In addition, PAPTs are essential for bacterial cell viability, HpPAPT can be a potential drug target for H. pylori by the complex structural information.

並列關鍵字

Spermidine Synthase

參考文獻

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