麩胱甘肽-精胺質水解酶在大腸桿菌氧化還原調控中所扮演的角色

Abstract

Glutathionylspermidne (Gsp) 是由glutathione和spermidine以醯胺鍵 (amide bond) 鍵結而形成的化合物。由於Gsp只存在於單細胞原蟲寄生蟲及大腸桿菌等菌體中，故與Gsp代謝相關的酵素被視為抗寄生蟲藥物發展的目標。Glutathionylspermidine synthetase/amidase (GspSA) 是一個雙功能性的酵素，它同時具有glutathionylspermidine synthetase (Gsp synthetase) 和glutathionylspermidine amidase (Gsp amidase) 兩個功能性區塊 (functional domain)，分別具有生成和降解Gsp的能力 (如下圖所示)。雖然大約13年以前就已經發現在大腸桿菌中具有GspSA的存在，但究竟GspSA在大腸桿菌中是扮演什麼角色？Gsp這個分子在大腸桿菌中具有什麼樣的功能？以及GspSA在菌體中是如何調控它兩個相反活性的功能區塊？一直以來都還沒有定論。 在先前的研究中發現在過氧化氫 (hydrogen peroxide, H2O2) 的處理下，GspSA會發生選擇性抑制，即Gsp amidase會因為active site中的cysteine-59受氧化而失去活性，但Gsp synthetase的活性不受影響。為了探討GspSA兩個功能區塊的調控方式，和瞭解如何調控Gsp的濃度，本論文建立了一套能靈敏偵測含硫醇 (thiol) 之分子的分析方法。先使用monobromobimane (mBBr) 與thiol分子進行衍生化，產生具有螢光之產物後，再利用HPLC進行對thiol分子的定性與定量分析。 利用此方法，我們發現在in vitro或in vivo的實驗中，過氧化氫的處理下都會造成Gsp amidase的選擇性抑制，並且導致Gsp快速而大量的累積，在此同時菌體中GspSA的蛋白質表現量則維持固定。此外我們也發現在營養不足及厭氧的條件下菌體中的Gsp也會有累積的情況發生。 最後，我們提出一個模式 (model) 藉以解釋在氧化壓力之下，大腸桿菌如何透過GspSA 調控菌體內的氧化還原平衡；此模式除了指出Gsp amidase的選擇性抑制會造成Gsp的累積，同時也解釋了Gsp disulfide (一種Gsp的氧化形式) 可以藉由Gsp amidase及GSH reductase兩種酵素的協同作用，而得以回復成GSH及spermidine，進而達成生理環境下thiol的平衡。

Glutathionylspermidine (Gsp), the conjugate of glutathione and spermidine, only appears in some parasitical protozoa or bacteria such as Escherichia coli. Therefore, the enzymes involved in Gsp metabolism are considered as the targets for anti-parasitic drugs. Glutathionylspermidine synthetase/amidase (GspSA) is a bifunctional enzyme to catalyze the biosynthesis and hydrolysis of Gsp shown as follows. Although GspSA was identified in Escharichia coli more than a decade ago, several issues still remain ambiguous. For instance, the physiological functions of Gsp and GspSA in E. coli are not clear. There is no clear answer regarding to how the two opposite activities of GspSA (Gsp synthetase and Gsp amidase) communicate. In the previous study, Gsp amidase was found to be selectively inactivated in the presence of hydrogen peroxide (H2O2) owing to temporary oxidation of cysteine-59 in the active site. In contrast, the activity of Gsp synthetase remained intact. In order to understand the regulation of GspSA and the Gsp turnover, a sensitive method was thus developed for monitoring the amount of small thiols by derivatization with mBBr, followed by HPLC analysis. In the in vitro and in vivo study, the Gsp level was found to be accumulative due to selective inactivation of Gsp amidase by H¬2O2, in contrast to the constant expression level of GspSA. In addition, Gsp was also found to increase under starvation or anaerobic conditions. A GspSA-based model was proposed to interpret how E. coli regulates the intracellular redox balance under oxidative stress. In addition to explaining how selective inactivation of Gsp amidase leading to the accumulation of Gsp, the model also demonstrates that Gsp disulfide, the oxidative form of Gsp, could be regenerated to GSH and spermidine by the Gsp amidase and GSH reductase couple.