碩士 / 國立陽明大學 / 生命科學系暨基因體科學研究所 / 105 / Trehalose synthase (TS) catalyzes the reversible conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis and hence has become an attractive candidate for industrial applications. Previously crystal structures of Deinococcus radiodurans TS (DrTS) in complex with the Tris inhibitor, represents a substrate-induced closed conformation for catalysis of the intramolecular isomerization. A detailed comparison with TS structural neighbors suggests that the diverse residues at the +1 subsite is responsible for the substrate and reaction specificity. Based on the DrTS–maltose modelled structure, replacement of Tyr213, E320, and E324 with alanine eliminates the enzyme activity, revealing the essentiality of these three residues. However, the N253A mutant displays a decreased isomerase activity and an increased hydrolase activity, suggesting that Asn253 is involved in the reaction specificity.
In this thesis, I first solved the complex structure of DrTS with validoxylamine A, a trehalose analogue. In addition, the modelled structures of DrTS in complex with maltose, sucrose and isomaltose were built. These structures and previous studies suggest an important role of Asn253 in the substrate-induced closed conformation and the reaction specificity. Therefore, saturated mutagenesis of N253 was carried out. The inactive N253R structure demonstrates that Arg253 protrudes into the +1 subsite and blocks substrate binding. In addition, four variants, N253A/C/D/L, possessed a reduced isomerase and an increased hydrolase activity, which may be explained by mutation induction of an active-site aperture for water entry, as that in the N253A and N253C structures. Moreover, the fourteen mutants, (N253E, N253F, N253G, N253H, N253I, N253K, N253M, N253P, N253Q, N253S, N253T, N253V, N253W and N253Y), retained only the maltase activity. The N253E and N253Q structures display that the side chain makes steric hindrance with the glucose of trehalose at the +1 subsite and hence trehalose can serve as neither substrate nor product. Finally, an empty active site is unexpectedly observed in the N253F/R structures, which may represent an open conformation for the apo enzyme prior to substrate binding. In summary, the TS-unique asparagine, Asn253 in DrTS, not only participates in the substrate-induced active-site closure but also is involved in the substrate and reaction specificity.
| Identifer | oai:union.ndltd.org:TW/105YM005105008 |
| Date | January 2017 |
| Creators | Yong-Jun Wei, 尉詠竣 |
| 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 | 66 |
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