幽門螺旋桿菌磷酸泛酸醯基乙胺腺苷轉移酶結構與功能之研究

Structural and Functional Studies of Phosphopantetheine aenylyltransferase from Helicobacter pylori

陳志豪(Chih-Hao Chen)

指導教授 ： 殷献生

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(2009年)

摘要

世界上估計將近有一半的人口都被幽門螺旋桿菌寄生著，它生活在人體的胃部及十二指腸。感染幽門螺旋桿菌會引發胃及十二指腸慢性發炎，長期的發炎會導致潰瘍的產生。由於抗生素的頻繁使用，對於抑制幽門螺旋桿菌已經造成效力減弱的結果。因此在抑制的機制上轉為研究專門對抗其生理代謝路線的方法。輔酶A是在所有生物的代謝上都會到用的醯基攜帶者，它牽涉到接近5%的生理代謝反應。在前人的研究發現抑制輔酶A的生合成可以有效達到抑制細菌生長的效果。輔酶A的生合成包含著五個步驟，其中倒數第二個步驟是一個速率決定步驟。這個步驟由酵素磷酸泛酸醯基乙胺腺苷轉移酶 (phosphopantetheine adenylyltransferas) (PPAT) 催化，並且會被下游合成的輔酶A來抑制其活性。PPAT在細菌裡有著很高的序列相似性，但是與人類相比卻沒有顯著的相似序列，因此發展對抗此酵素的藥物對於抑制幽門螺旋桿菌有很大的幫助。在研究此酵素的結構上使用了X-光結晶學的方法，得到了幽門螺旋桿菌PPAT的晶體，並且解出其結構。這個結構是一個open form 的構形，這是在其他物種的PPAT上沒見過的。本文使用圓二色光譜來探討PPAT的二級結構的穩定性，另外也使用了恒溫滴定熱卡計來分析其酵素催化能力。

關鍵字

幽門螺旋桿菌 ； 輔酶A ； 磷酸泛酸醯基乙胺腺苷轉移酶

並列摘要

Coenzyme A (CoA) is the principle acyl carrier in all living cells, and is required for many metabolic reactions, including citric acid cycle and fatty acid metabolism. Phosphopantetheine adenylyltransferase (PPAT; EC: 2.7.7.3) is an essential enzyme that catalyzes the penultimate step of CoA biosynthesis by transferring an adenylyl group from ATP to 4’-phosphopantetheine (Ppant), yielding 3’-dephospho-CoA (dPCoA) and pyrophosphate. PPAT catalyzes the second rate-limiting step in CoA synthetic pathway and is regulated by feedback inhibition by downstream end product, CoA. The bacteria PPAT sequences show high degree of homology but are dissimilar from mammalian, therefore, this enzyme might be a good antibacterial target. PPAT from Helicobacter pylori (H. pylori) which infects about 50% world population and cause gastric and duodenal ulcers was expressed and crystallized in space group I222. The crystal structure of H. pylori PPAT has been determined by molecular replacement method at 1.75 Å resolution. The structure shows an open form of H. pylori PPAT that have not been observed in other PPATs. Since previous reports have shown that PPAT from Escherichia coli exhibites pH-dependent binding affinity towards substrates, the PPAT structural stability and activity was studied by circular dichroism and isothermal titration calorimetry.

並列關鍵字

Helicobacter pylori ； Coenzyme A ； Phosphopantetheine adenylyltransferase

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