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Diversity-generating mechanisms of non-ribosomal peptide synthetases :I. Enzymes in the biosynthesis of 3,5-dihydroxy-L-phenylglycine. II. Thioesterase domain of the surfactin synthetase

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Diversity-generating mechanisms of non-ribosomal peptide synthetases :
其他題名:	I. Enzymes in the biosynthesis of 3,5-dihydroxy-L-phenylglycine. II. Thioesterase domain of the surfactin synthetase
作者:	Tseng, Claire Chia-Hui.
面頁冊數:	132 p.
附註:	Adviser: Christopher T. Walsh.
附註:	Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2406.
Contained By:	Dissertation Abstracts International65-05B.
標題:	Chemistry, Biochemistry.
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3132012
ISBN:	0496792520

Diversity-generating mechanisms of non-ribosomal peptide synthetases :I. Enzymes in the biosynthesis of 3,5-dihydroxy-L-phenylglycine. II. Thioesterase domain of the surfactin synthetase
Tseng, Claire Chia-Hui.

Diversity-generating mechanisms of non-ribosomal peptide synthetases :I. Enzymes in the biosynthesis of 3,5-dihydroxy-L-phenylglycine. II. Thioesterase domain of the surfactin synthetase [electronic resource] - 132 p.

Adviser: Christopher T. Walsh.

Thesis (Ph.D.)--Harvard University, 2004.

The TEs of NRPSs are responsible for the release of the synthesized linear peptide from the synthetase through either hydrolysis or cyclization. The TE of the surfactin synthetase catalyzes the cyclization of a linear lipoheptapeptide through the formation of an ester linkage between the C-terminal amino acid and the hydroxyl group of a beta-hydroxy fatty acid attached to the N-terminal amino acid. By applying kinetic analysis with the systematic synthesis of substrate variants and enzymatic mutagenesis based on structural information, we characterized how NRPSs achieve distinctive macrocyclic structure using the surfactin TE. Based upon this biochemical data and complementary cocrystal structural studies, the cyclizing conformation of the surfactin peptide was modeled into the TE active site.

ISBN: 0496792520Subjects--Topical Terms:

226900
Chemistry, Biochemistry.

Diversity-generating mechanisms of non-ribosomal peptide synthetases :I. Enzymes in the biosynthesis of 3,5-dihydroxy-L-phenylglycine. II. Thioesterase domain of the surfactin synthetase
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520 # $a The TEs of NRPSs are responsible for the release of the synthesized linear peptide from the synthetase through either hydrolysis or cyclization. The TE of the surfactin synthetase catalyzes the cyclization of a linear lipoheptapeptide through the formation of an ester linkage between the C-terminal amino acid and the hydroxyl group of a beta-hydroxy fatty acid attached to the N-terminal amino acid. By applying kinetic analysis with the systematic synthesis of substrate variants and enzymatic mutagenesis based on structural information, we characterized how NRPSs achieve distinctive macrocyclic structure using the surfactin TE. Based upon this biochemical data and complementary cocrystal structural studies, the cyclizing conformation of the surfactin peptide was modeled into the TE active site.
520 # $a This thesis examines the mechanistic enzymology of two aspects of nonribosomal peptide synthetases (NRPSs), which use unique methods to produce medically important and structurally intriguing peptidic molecules: (1) the biosynthetic pathway of 3,5-dihydroxy-L-phenylglycine (Dpg), a non-proteinogenic amino acid used in the biosynthesis of the vancomycin and teicoplanin groups of antibiotics; and (2) the thioesterase domain (TE) of the synthetase responsible for the synthesis of surfactin, an antibiotic with powerful surfactant properties.
520 # $a We elucidated the framework for the biosynthetic pathway of 3,5-dihydroxyphenylglyoxylate (DPGx), the precursor to Dpg by a transamination, reconstituting its biosynthesis in vitro. We characterized the enzymatic activities and specificities of the four proteins, DpgA-D, in the pathway, and further investigated the mechanisms of action of DpgA and DpgC, revealing novel enzymatic activities in each. DpgA, a type III polyketide synthase, catalyzes the decarboxylative condensation and cyclization of four malonyl-coenzyme A (CoA) molecules to 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA), with the aid of DpgB and DpgD. DpgC is a remarkable cofactor-less dioxygenase that then oxidizes the alpha-position of DPA-CoA to a keto group and cleaves the CoA-thioester, to form DPGx. We employed a variety of approaches to probe these unique mechanisms of action, including the use of active site mutants and substrate labeling techniques, as well as the synthesis and characterization of intermediates and alternate substrates.
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