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首页> 外文学位 >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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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.

机译：非核糖体肽合成酶的多样性产生机制：I. 3,5-二羟基-L-苯基甘氨酸生物合成中的酶。二。表面活性素合成酶的硫酯酶结构域。

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摘要

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.; 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.; 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.
机译：本文研究了非核糖体肽合成酶（NRPSs）两个方面的机械酶学，这两个方面使用独特的方法来生产医学上重要且结构上令人着迷的肽类分子：（1）3,5-二羟基-L-苯基甘氨酸（Dpg）的生物合成途径，用于万古霉素和替考拉宁类抗生素生物合成的非蛋白氨基酸； （2）负责合成表面活性剂（一种具有强大表面活性剂特性的抗生素）的合成酶的硫酯酶结构域（TE）。我们阐明了3,5-二羟基苯基乙醛酸酯（DPGx）的生物合成途径的框架，这是通过转氨作用形成Dpg的前体，并在体外重建了其生物合成。我们表征了该途径中四种蛋白质DpgA-D的酶促活性和特异性，并进一步研究了DpgA和DpgC的作用机理，揭示了每种中的新型酶促活性。 DpgA是一种III型聚酮化合物合酶，借助DpgB和DpgD催化四个丙二酰辅酶A（CoA）分子脱羧缩合和环化为3,5-二羟基苯基乙酰基-CoA（DPA-CoA）。 DpgC是一种出色的无辅因子双加氧酶，它随后将DPA-CoA的α-位氧化为酮基并裂解CoA-硫酯，形成DPGx。我们采用了多种方法来探究这些独特的作用机理，包括使用活性位点突变体和底物标记技术，以及中间体和替代底物的合成和表征。 NRPS的TE负责通过水解或环化作用从合成酶释放合成的线性肽。表面活性素合成酶的TE通过在C末端氨基酸和连接至N末端氨基酸的β-羟基脂肪酸的羟基之间形成酯键来催化线性脂庚肽的环化。通过应用动力学分析与底物变体的系统合成以及基于结构信息的酶促诱变，我们表征了NRPS如何使用表面活性素TE实现独特的大环结构。基于此生化数据和互补的共晶体结构研究，将表面活性素肽的环构象建模到TE活性位点中。

著录项

  • 作者

    Tseng, Claire Chia-Hui.;

  • 作者单位

    Harvard University.;

  • 授予单位 Harvard University.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 132 p.
  • 总页数 132
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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