博士 / 國立陽明大學 / 生化暨分子生物研究所 / 103 / An intracellular poly-3-hydroxybutyrate depolymerase from Bacillus thuringiensis (BtPhaZ) and a trehalose synthase from Deinococcus radiodurans (DrTS) have been screened for potential inductrial applications in polyester biodegradation and trehalose production, respectively. In this dissertation, I first determined the crystal structures of these two enzymes by the X-ray diffraction method and then performed structural-based mutational analysis in order to understand the ezyme mechanism and substrate specifity. Hopefully, my studies would provide useful information for development of more efficient enzymes for potential industrial applications.
BtPhaZ catalyzes the degradation of poly-3-hydroxybutyrate into 3-hydroxy- butyrate (3HB). The recombinant protein has been crystallized and the X-ray diffraction data have been collected at 1.42 Å resolution by Chen Chia-Lin. Here I solved the BtPhaZ structure using the molecular replacement method even BtPhaZ only shares 24% sequence identity to the proteins in the PDB database. BtPhaZ consists of a canonical /hydrolase fold and a helical domain. A detailed structural comparison reveals three new conserved signatures, HG36, D61xxGxG and G248xxD, in addition to the most conserved signature in the /hydrolase superfamily, GxS102xG. Three mutants including G36A, D61A and G248A were generated and characterized. The turbidimetric assay revealed that G36A and G248A displayed 5% and 23% activities of the wild type, respectively. The esterase activity assay showed that G36A displayed a 10,000-fold decrease in kcat compared to the wild type. The decreased activities of G36A and G248A may be due to unfavorable contacts with surrounding residues such as Trp101 and Cys277, respectively. The D61A mutant was expressed in the inclusion body, suggesting that the extensive interactions between Asp61and Ser40, Ser41, and Asn67 are essential for the structural integrity. Therefore, these four conserved signatures not only constitute the catalytic triad and the oxyanion hole, but also attain the active-site conformation. In addition, a 3HB trimer was modeled into the active site with subsequent mutational analysis. The turbidimetric assay revealed that T39V, Y133F, Y133A and N214A showed a similar hydrolytic activity to the wild type, while N37A and N37D retained 20% activity. Activity assays were consistent with the complex model, in which Asn37Nwas proposed to interact with the carbonyl group of the 3HB at the +2 stie. Moreover, a cluster of exposed hydrophobic residues may be responsible for the PHB attachment, including Val146, Leu149, Val161, Leu176, Leu184, Tyr252, Val253 and Val257. The L176E, L184E and Y252E mutants were first generated, and the turbidimetric assay revealed that L184E displayed 15% activity of the wild type, thus Leu184 is important for the PHB attachment. Furthermore, a putative fragment containing seven units of ethylene glycol was observed at the active site, which forms hydrophobic contacts with Met38, Trp101, Met103, Tyr133, Val166, Tyr177, Val181, Trp182, Leu184, Leu185, Ile186 and Val253. In the future, more mutants will be generated to approach the polymer binding, and hopefully, the mutants with higher PHB affinities will be obtained.
DrTS catalyzes the conversion of the inexpensive maltose to trehalose with high substrate specificity and high conversion rates. The recombinant protein has been crystallized by Lin Yi-Ting and the X-ray diffraction data have been collected at 2.7 Å resolution. I determined the dimeric DrTS-Tris structure by the molecular replacement method. DrTS belongs to glycoside hydrolase family 13, and consists of a catalytic (/)8 domain, subdomain B, domain C and two TS-unique subdomains (S7 and S8). The domain C and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase, and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The interaction networks between subdomain B and S7 seal the active-site entrance that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 89-fold and an increased hydrolase activity by 1.51.8-fold. The N253A structure showed a small pore created for water entry. Interestingly, the nonreducing terminal maltosyl residue in the NpAS-maltoheptoase complex fits the -1 and +1 site in DrTS-Tris well, and hence a DrTS-maltose structure was modeled. This model suggested the Tyr213, Glu320 and Glu324 form hydrogen bonds to the glucose unit at the +1 site. Neither isomerase nor hydrolase activity was detected when the Y213A, E320A and E324A mutants wre examed. Finally, the DrTS-maltose structure suggested that some residues are involed in substrate and reation specificity. Also modification of several residues may result in formation of more stable tetramer. Hopefully, we can gain some mutants with high catalytic efficiency and high thermostability.
| Identifer | oai:union.ndltd.org:TW/103YM005107011 |
| Date | January 2015 |
| Creators | Yung-Lin Wang, 王詠霖 |
| 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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