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Engineering and analysis of 6-deoxye...

Chen, Yu-Chieh Alice.

Engineering and analysis of 6-deoxyerythronolide B synthase.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Engineering and analysis of 6-deoxyerythronolide B synthase.
作者:	Chen, Yu-Chieh Alice.
面頁冊數:	127 p.
附註:	Adviser: Chaitan Khosla.
附註:	Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 3140.
Contained By:	Dissertation Abstracts International69-05B.
標題:	Chemistry, Biochemistry.
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3313552
ISBN:	9780549623892

Engineering and analysis of 6-deoxyerythronolide B synthase.
Chen, Yu-Chieh Alice.

Engineering and analysis of 6-deoxyerythronolide B synthase. - 127 p.

Adviser: Chaitan Khosla.

Thesis (Ph.D.)--Stanford University, 2008.

Combining the aforementioned studies, it appears that the [KS][AT]--ACP pairing is the most specific among all the domain interactions between [KS] [AT], ketoreductase (KR), and ACP. To understand this potential rate-limiting interaction, we have attempted to identify the ACP residue(s) involved in [KS] [AT]---ACP recognition through a series of ACP mutagenesis and protein fusion of DEBS ACP3 and ACP6. It has been found that loop I of an ACP may determine its [KS] [AT] specificity, while helix II of an ACP plays a significant role in structural integrity.

ISBN: 9780549623892Subjects--Topical Terms:

226900
Chemistry, Biochemistry.

Engineering and analysis of 6-deoxyerythronolide B synthase.
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100 $a Chen, Yu-Chieh Alice. $3 321821
245 1 0 $a Engineering and analysis of 6-deoxyerythronolide B synthase.
300 $a 127 p.
500 $a Adviser: Chaitan Khosla.
500 $a Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 3140.
502 $a Thesis (Ph.D.)--Stanford University, 2008.
520 $a Combining the aforementioned studies, it appears that the [KS][AT]--ACP pairing is the most specific among all the domain interactions between [KS] [AT], ketoreductase (KR), and ACP. To understand this potential rate-limiting interaction, we have attempted to identify the ACP residue(s) involved in [KS] [AT]---ACP recognition through a series of ACP mutagenesis and protein fusion of DEBS ACP3 and ACP6. It has been found that loop I of an ACP may determine its [KS] [AT] specificity, while helix II of an ACP plays a significant role in structural integrity.
520 $a Coupled with mechanistic and structural insights derived from recent studies, we anticipate improved capability of designing efficient hybrid PKS systems to produce novel, medicinally potent polyketide compounds through biosynthetic pathways.
520 $a Despite the engineering-friendly organization, modular PKSs are well known for their structural complexity, a feature that prevents detailed mechanistic studies of individual catalytic domains. Consequently, the efficiency of many PKS engineering strategies have been compromised by factors such as impaired protein-protein interaction between domains, suboptimal domain junctions employed in engineering, and the enzymes' intrinsic intolerance for unnatural substrates.
520 $a Lastly, building upon the domain activities established in the fragmentation studies, this report subsequently explores different PKS configurations for effective introduction of polyketide chemical diversity. In particular, we have examined the intermolecular (in trans) complementation of ketoreductase and acyltransferase domains in a modular context. The studies show that a KR domain from tylactone synthase (TYLS) and the AT domain from disorazole synthase (DSZS) are capable of interacting with DEBS modules to produce ketide products unnatural to the DEBS system. Unlike strategies involving extensive domain or module modifications, this alternative approach minimizes structural perturbation to the PKS system and allows convenient permutations for the incorporation of unnatural functionalities.
520 $a Modular polyketide synthases (PKSs) are enzymatic assembly lines that catalyze the production of polyketide compounds, many of which exhibit antibiotic, antifungal, anticancer, and other important biological activities. Individual modules of a modular PKS such as 6- deoxyerythronolide B synthase (DEBS) consist of highly conserved catalytic domains. The domains from a given module cooperate to synthesize polyketides from simple acyl-CoA precursors via a thiotemplate mechanism similar to that of fatty acid synthesis. The co-linearity between the organization of a PKS and the structure of the corresponding product has made PKS genetic engineering an emerging strategy for polyketide analog generation.
520 $a Specifically, we have constructed fully functional ketosynthase-acyltransferase ([KS][AT]) di-domains from DEBS modules to facilitate the interrogation into protein-protein interactions between intramodular domains. The pronounced specificity between [KS] [AT] and acyl carrier protein (ACP) demonstrates that domains derived from different modules are not always functionally compatible, highlighting the importance of domain pairing choices in constructing hybrid PKSs. The further dissection and reconstitution of a module also provide a platform for defining authentic domain boundaries and examining contributions of each domain to substrate specificity, catalytic activity, and product stereocontrol, most of which would have remained unexplored in intact module systems. Based on the kinetic insights, a productive hybrid module [KS3][AT3][KR5][ACP5][TE] has been constructed. The high expression level and functionality of this hybrid protein demonstrate the usefulness of kinetic and structural analyses for hybrid module design.
520 $a To unravel the inherent complexity of PKSs, studies in this thesis work to dissect the DEBS assembly line into its constituent subunits through protein engineering. This "divide and conquer" approach has allowed us to construct numerous isolated subunits for in-depth functional and structural analyses. Through these studies, a set of engineering guidelines for heterologous PKS construction is proposed in the following areas: (a) authentic domain boundaries, (b) domain-domain compatibilities, (c) relative importance of domain-domain interactions, and (d) feasibility of alternative PKS configurations.
590 $a School code: 0212.
650 $a Chemistry, Biochemistry. $3 226900
650 $a Engineering, Chemical. $3 226989
690 $a 0487
690 $a 0542
710 $a Stanford University. $3 212607
773 0 $g 69-05B. $t Dissertation Abstracts International
790 $a 0212
790 1 0 $a Khosla, Chaitan, $e advisor
791 $a Ph.D.
792 $a 2008
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