Understanding the substrate specificity of sulfonucleotide reductases based on structural analysis and newly developed non-radioactive assay
| Autor: | Chia-Wei Chou, 周家緯 |
|---|---|
| Rok vydání: | 2016 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 104 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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