摘要:細菌、真菌及植物進行生合成含硫化合物時,會由外界取得的硫酸根,進行一連串還原代謝固硫作用獲得硫元素,SNR(sulfonucleotide reductase)為此代謝途徑中一重要的關鍵酵素。而根據受質的差異,又可分為PAPR(PAPS reductase)與APR(APS reductase)。此類酵素之催化反應為兩階段機制,先以酵素尾端Cysteine之thiol group對硫酸核苷進行親核性攻擊,而形成Cysteine上的thiosulfonate中間體,之後再由thioredoxin等還原蛋白將其還原,並生成亞硫酸根與AMP或PAP。而植物的APR酵素於C端比一般APR多了一個thioredoxin功能區塊,更顯現可能有著對向調節或合作的四級結構存在。由於此類酵素的催化反應涉及許多步驟,其運行機制仍然有許多未知的部份尚待釐清。值得注意的是,哺乳動物並不會進行此類的還原途徑,這也使得病原菌的此類酵素被視為新穎抗病菌藥物開發的潛力目標。
為進一步認識硫酸腺苷還原酵素家族其蛋白結構與功能間的相關性,我們選擇由極端嗜熱硫化葉菌之耐熱APR酵素作為研究模型,因為耐熱酵素通常在室溫下運作較為緩慢,因此有機會能在常溫觀察到更精確的作用機制。而植物APR酵素為另一種有趣的目標,目前沒有任何有關植物APR三級與四級結構的相關議題被研究。在本計畫,我們希望從事以下的探討:1) 解析古生菌與植物APR酵素的原子層級立體結構與催化活性中心◦ 2) 分析及比較APS與PAPS受質進入APR酵素結合區的構型與胺基酸組成喜好差異◦ 3) 瞭解此酵素受thioredoxin調節造成達成尾端活性區構型變化之分子機制◦ 4) 解構APR酵素的催化機制與保守胺基酸作用與重要性◦ 5) 深入探討植物APR的區塊架構結構與合作模式◦為達成這些目標,我們已成功解析APR酵素thiosulfonate中間體與AMP結合的蛋白晶體結構◦此外,我們也已獲得了柳化葉菌之兩個thioredoxin蛋白與thioredoxin還原酶。我們亦已構築或取得植物APR酵素的表達載體及數個APR之突變株。在取得高純度的蛋白樣本後,除了解析蛋白自身的結構外,也將從事酵素與受質類似物或抑制劑形成之複合體的結構解析◦基於這些豐富的初期結果,上述之各個研究主題應能順利達成◦
Abstract: Assimilatory sulfate reduction supplies prototrophic organisms (such as plants, fungi, and many bacteria) with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation via an ATP-dependent activation to form adenosine-5′-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes are collectively known as sulfonucleotide reductases (SNRs). In a two-step mechanism, the sulfonucleotide undergoes nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S–SO3–) intermediate, followed by release of sulfite in a thioredoxin-dependent manner. Interestingly, Plant APR contains an extended C-terminal domain, which shares structural and functional similarity to thioredoxin. It may possess a more efficient “trans” regulation or cooperative mechanism for this “chimera”. Since the complexity of multi-step reaction, the detailed catalytic mechanism of SNR remains elusive. Notably, mammals do not possess the sulfate reduction pathway, which makes SNR in pathogenic bacteria being a promising target for drug development against human pathogens.
To provide a better understanding on the structure/function relationship of APR enzymes family, we choose thermostable APR from Sulfolobus solfataricus as a model. Because thermophillic enzymes evolved to function at high temperatures, they tend to function more slowly at room temperature and are therefore excellent mechanistic models. In addition, another interesting target goes on plant APR, since there is little known information of their structural architectures. In this proposal, we would like to engage in the following studies: 1) obtain atomic resolution pictures of overall structural architecture and the active sites of archaeal APR and plant APR enzymes, 2) compare the substrate-recognition differences between APS and PAPS for APR enzymes, 3) understand the structural basis of thioredoxin-dependent tail springing mechanism of APR, 4) decipher the roles of conserved residues in the catalytic mechanisms of APR enzymes. 5) delineate the overall structural architecture and cooperative mechanism of plant APR. Towards these goals, we have successfully determined a high resolution crystal structure of thiosulfonate intermediate and AMP-bound forms of APR recently. In addition, we have constructed/obtained expression plasmids for expressing soluble forms of thioredoxin ssoA1 and ssoA2, and thioredoxin reductase SsoB1 from Sulfolobus sulfataricus, as well as AtAPR1, AtAPR2 and AtAPR3 from Arabidopsis thaliana. After purified proteins are available, various binary complexes formed between enzyme(s) and substrate analog(s)/product(s) as well regulatory protein(s) will be used for crystallization. Moreover, a series of mutant enzymes with altered catalytic behavior has been identified and their structures will also be examined. On the basis of our solid preliminary results, it is expected that significant progress toward understanding the structure/functional relationship of APR enzymes family will be achieved.