碩士 / 國立陽明大學 / 生命科學系暨基因體科學研究所 / 102 / The disaccharide trehalose displays exceptional stability and has been used in the food, cosmetic, and biopharmaceutical industries. Trehalose synthase catalyzes a simple conversion of the inexpensive maltose into trehalose and hence has a great industrial potential. A trehalose synthase from Deinococcus radiodurans (DrTS) has been screened for potential applications in trehalose production. However, the disadvantages of low thermo-stability and the side product of glucose by the hydrolytic activity are needed to be improved. In this thesis, I perform structural and functional studies on DrTS for a better understanding of its catalytic mechanism.
The DrTS structure has been determined at 2.7Å-resolution by Wang Yung-Lin in the lab. DrTS consists of a catalytic (/)8 barrel, a subdomain B, a C-terminal domain and two TS-unique subdomains (S7 and S8). The inhibitor Tris molecule is bound at the P-1 site and interacts with several conserved residues as other glycoside hydrolase 13 (GH13) members. In this thesis, maltose was modeled into the active site of DrTS. A detailed structural comparison with sucrose isomerase and sucrose hydrolase was carried out to identify the residues involved in isomerase/hydrolase activities. Several TS-unique, for example residues Ile150 and Asn253 are predicted to involve in the substrate and reaction specificity, while Arg316 in the active site stability.
Five mutants including I150F, R316C, N253E, N253F and N253Q, were then generated and charactenized. The activity assay revealed that I150F displayed a half of activity to wild type. R316C showed a half of hydrolylase activity, but had an isomerase activity of ~56%. However, N253E and N253Q retained the 75 and 53 % hydrolase activity without any detectable isomerase activity, whereas N253F displayed no detectable activity for both isomerization and hydrolysis. In addition, I obtained crystals from R316C and N253F, and collected the X-ray diffraction data at NSRRC. The crystal structures were determined at 2.7 and 2.85 Å, respectively. The overall structure of R316C displayed similar closed comformation to the wild type structure (a RMSD of 0.29 Å for 548 C). Unexpectedly, the N253F revealed an inactive open conformation (a RMSD 1.15 Å for 508 C), which may represent an apo-form structure prior the substrate binding. These results may give us a better understanding of detailed mechanisms of substrate and reaction specificity for TS, which could help us to further manipulate this important industrial enzyme.
| Identifer | oai:union.ndltd.org:TW/102YM005105031 |
| Date | January 2014 |
| Creators | Yu-Chiao Hsieh, 謝宇喬 |
| Contributors | Shwu-Huey Liaw, 廖淑惠 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 59 |
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