以結構分析與新發展之非放射性物質的酵素活性測定方法探討硫酸腺苷還原酶的受質選擇性

硫酸腺苷還原酶 (sulfonucleotide reductase ; SNR) ，為細菌、真菌及植物生合成生存必需之含硫化合物的系列反應中，將外界取得的 sulfate (SO42-) 進行硫同化 (sulfate assimilation)之關鍵反應步驟酵素。由於哺乳動物並不會進行此類的還原途徑，使得此酵素被視為新穎抗菌藥物開發的潛力目標，因此SNR的快速活性測試方法對於藥物的篩選有其必要性。在過去僅有兩種方法被使用於此酵素的活性測定，而且這兩種方法都必須使用到放射性元素標定，這些放射性方法最主要的缺點就是必須特別注意這些放射性元素的物質與廢棄物。本研究發展一新穎的非放射性酵素活性測定法，希望此簡單且價格低廉的比色法可有利於高通量的藥物篩選。我們利用枯草桿菌的硫酸腺苷還原酶 (sulfonucleotide reductase from Bacillus subtilis, BsSNR) 進行研究與方法的開發，此一酵素有趣的是其兼具有 APS (adenosine 5’phosphosulfate) reductase 與 PAPS(3’phosphoadenosine 5’phosphosulfate) reductase 的活性，兩種基質皆能被催化。經由表現純化後所得到的重組蛋白呈現棕色來看，暗示了蛋白中可能含有金屬錯化物基團在其中，再藉由 UV-Vis 吸光光譜和鐵硫簇移除試驗，我們可以推測 [4Fe-4S] 簇輔基團的存在。此外，利用圓二色光譜儀 (circular dichroism) 的測定可以得知此種蛋白的 Tm 約在 56.1°C 左右。為獲得更詳盡的結構資訊，我們也利用已知結構作為模版對 BsSNR 進行結構模擬分析，藉由比較 BsSNR-APS 與 BsSNR-PAPS 兩種不同複合物結構，我們發現到數個參與催化活性過程中的重要殘基並且推測出其功能。根據本篇實驗的結果，我們提供了一個新的酵素活性測定方法，此一方法可以透過比色法的方式測定不論 PAPS 或者 APS 經由 SNR 反應後產生的亞硫酸根。我們也利用此方法測定 BsSNR 的酵素動力學參數，並且比較放射性元素標定方法之酵素動力學參數，這些結果顯示此方法於 SNR 相關的酵素研究與藥物篩選應用有一定的可靠性。

關鍵字

枯草桿菌；磷酸腺苷還原酶；活性測定

並列摘要

Sulfonucleotide reductase (SNR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of bacterial infection. However, metazoans do not possess the sulfate reduction pathway, which makes sulfonucleotide reductases being a promising target for drug development against human pathogens. As such, a rapid assay of SNR activity is valuable in drug screening. Only two methods are currently used to measure SNR activity, both involving radiolabeled material, however, the chief disadvantage of these assays is that special precautions are required due to the radioactive material and wastes involved. In this study, we developed a novel non-radioactive assay for monitoring SNR activity, and would like to apply this siple and cost-effective colorimetric method for high-throughput drug screening. BsSNR (sulfonucleotide reductases from Bacillus subtilis) were used to develop the newly assay and study the substrates specificity of SNR, Interestingly, both PAPS (3'phosphoadenosine 5′-phosphosulfate) and APS (adenosine 5′-phosphosulfate) are substrates for BsSNR. Purified proteins in solution are shown brownish in color and proposed they should contain one [4Fe-4S] cluster per polypeptide chain. Data from ultraviolet-visible absorption spectroscopy would be collected to elucidate the nature of the prosthetic group containing properties. CD experiments showed that BsSNR possesses the Tm about 56.1 °C. To explain the substrates specificity, we got a homology modeling of BsSNR, and found the important residues involving in the binding of substrates. Taken together, we developed a novel SNR activity assay, which involves of either APS- or PAPS-dependent sulfite generation using colorimetric method. Based on the method, we determined the substrate selectivity and enzyme kinetic parameters of BsSNR. Our results reveal the current method is reliable with many potential applications in SNR studies and drug screening.

並列關鍵字

Bacillus subtilis ； sulfonucleotide reductase ； activity assay

參考文獻

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以結構分析與新發展之非放射性物質的酵素活性測定方法探討硫酸腺苷還原酶的受質選擇性

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