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。同時植物的此類酵素則有助於抗逆境調控,此研究也將提供轉殖植物增加耐受性之分子基礎。
在過去半年來執行前期一年期計畫結果,我們已經精修完成APR酵素thiosulfonate中間體與AMP結合的晶體結構,並將完成酵素的物理化學特性及生化特性解析。由於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。這些結果使我們可得知微生物APR之tail於binding pocket的結合模式,為第一次的詳盡闡述。而本三年計畫旨在以系統性地利用野生型、各突變株之微生物和植物APR配合添加各受質等方式,對於酵素的催化機制與基質辨識進行物理化學特性探索與結構闡述。此研究將延續我們建立的系統與結果提出了五個目標:
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[4Fe-4S]鐵硫簇與保守胺基酸於催化機制之作用與重要性◦ 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,我們已成功生產數種植物與微生物APR酵素蛋白並獲得部分蛋白質晶體。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◦此外如初步資料所示,配合數種物理化學分析方法, 如核磁共振光譜、量熱儀等測量,將可釐清酵素催化機制與基質辨識。X光吸收光譜、電子自旋共振光譜、圓二色光譜則可進一步瞭解鐵硫簇於酵素之角色,及與催化機制之關聯性。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,我們的初步資料也顯示其催化機制似乎伴隨著四級結構變化。基於這些豐富的初期結果與確切目標,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. In addition, the studies of plant APR may provide molecular basis for transgenic plant to enhance tolerance against environmental stress.
Implementation of the previous one-year project in the past six months, we have refined the crystal structures of APR enzymes in complex with AMP and the thiosulfonate intermediate. The physicochemical and biochemical characterizations of the enzyme also will be completed. Thermostable APR from Sulfolobus solfataricus was chosen 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. The solved crystal structural of APR reveals the binding mode between catalytic tail and binding pocket of enzyme. In this proposal, Catalytic mechanism and substrate recognition of microbial and plant APR would be systematically approaching with at least wild-type, truncated forms and cysteine/serine variants as well as addition of ligands via physicochemical characterization and structural elucidation. Therefore, 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 [4Fe-4S] cluster in the catalytic mechanisms of APR enzymes. 5) delineate the overall structural architecture and cooperative mechanism of plant APR.
Towards these goals, we have constructed/obtained expression plasmids for expressing soluble forms of various microbial and plant APRs, and several protein crystals’ conditions have been screened out. 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 by various physicochemical methods, such as NMR, ITC. The role of iron-sulfur cluster in catalytic mechanism of APR also will be investigated by using EXAFS, EPR and CD spectroscopy. In addition, another interesting target goes on plant APR, since there is little structural information available, while our preliminary data shows a quaternary structural assembly occurred in plant APR catalytic action. On the basis of our solid preliminary results and the certain approaches, it is expected that significant progress toward understanding the structure/functional relationship of APR enzymes family will be achieved.