碩士 / 國立陽明大學 / 生命科學系暨基因體科學研究所 / 101 / For potential industrial applications in trehalose production and polymer biodegradation, an intracellular a trehalose synthase from Deinococcus radiodurans (DrTS) and poly-3-hydroxybutyrate depolymerase from Bacillus thuringiensis (BtPhaZ) have been screened, respectively. In this thesis, I perform structural studies on these two enzymes for a better understanding of their mechanisms of substrate specificity and enzyme catalysis. For DrTS, protein crystals were grown and the dimeric structure was determined at 2.7 Å-resolution. DrTS consists of a catalytic (/)8 TIM barrel and C-terminal domain with three additional subdomains. The C-terminal domain contributes the majority of the dimeric interface. A detailed structural comparison reveals that virtually identical interaction networks in stabilization of the orientations of the catalytic triad were observed in DrTS, suggesting of an active conformation. Maltose and trehalose could then be modeled into the active site, showing the detailed interaction of the substrate recognition. The interaction networks formed between subdomain B、S7 and TIM barrel loops seal the active site entrance, revealing a close conformation. Then these additional subdomain are involved in switch of the open/close conformation. Such regulation mechanism by subdomains of conformational change during enzyme catalysis are also observed in other GH13 members. On the other hand, Mycobacterium smegmatis trehalose synthase (MsTS) display an inactive conformation because the S7 loop blocks the substrate-binding site and distinct interaction networks are formed.
For BtPhaZ, the crystal structure has been solved by Wang Yung-Lin in the lab, which is composed of a /hydrolase fold and a helical cap domain. Subsequently I carried out a detailed structural comparison, which suggested three new conserved signatures, HG36, D61xxGxG and G248xxD, in addition to the most conserved signature in the large /hydrolase superfamily, GxSxG. Three mutants were generated and characterized including G36A, D61A and G248A. The D61A mutant was expressed in the inclusion body, suggesting that the interactions between Asp61and Ser40, Ser41, and Asn67 are important for the structural integrity. The turbidietric assay revealed that G36A and G248A displayed 4 % and 30 % activity, respectively, compared to wild type, perhaps due to unfavorable contacts of Ala36 and Ala248 with Met103 and Cys277, respectively. The distances between Gly36C and Met130N, and Gly248C and Cys277O are 4.1 Å, and 3.2 Å. Therefore, these four signatures not only constitute the catalytic triad and the oxyanion hole, but also their extensive interaction networks attain the active-site conformation. In addition, a 3-hydroxybutyrate (3HB) trimer was modeled into the active site of BtPhaZ, in which residues surrounding the putative P-1, P1 and P2 subsites were predicted. Seven mutants were generated and characterized including N37A, N37D, T39V, Y133F, Y133A, N214A and N214D. The turbidimetric assay revealed that T39V, Y133F, Y133A and N214A showed a similar hydrolytic activity to wild type, while N37A retained 20 % activity. Interestingly, the N37D contained ~20 % activity at pH 7.5, ~10 % activity at pH 8.5, whereas no detectable activity at pH 9. A kinetic assay revealed that replacement of Asn37 with aspartate resulted in a decreased in a Km, but not kcat. The activity assays were consistent with the complex model, in which Asn37Nwas proposed to interact with the carbonyl group of the 3HB at the P2 substie. Finaly, a putative adsorption site for the polymer substrate is formed by a clouster of exposed hydrophobic residues on the cap domain, including Val146、Leu149、Val161、Leu176、Leu184、Tyr252、Val253 and Val257.
| Identifer | oai:union.ndltd.org:TW/101YM005105045 |
| Date | January 2013 |
| Creators | Yi-Ting Lin, 林易霆 |
| 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 | 93 |
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