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首页> 外文学位 >Enzymatic characterization of Escherichia coli methylerythritol phosphate synthase and synthesis of methylerythritol phosphate pathway metabolites.
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Enzymatic characterization of Escherichia coli methylerythritol phosphate synthase and synthesis of methylerythritol phosphate pathway metabolites.

机译：大肠杆菌的甲基赤藓糖醇磷酸合酶的酶学表征和甲基赤藓糖醇磷酸途径代谢物的合成。

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摘要

The methylerythritol phosphate (MEP) pathway has been identified as a novel route to isoprenoid compounds operating in bacteria, algae, and plant chloroplasts. MEP synthase, which catalyzes the rearrangement/reduction of 1-deoxy-D-xylulose-5-phosphate (DXP) to form 2-C-methyl- D-erythritol-4-phosphate (MEP), is a key step in the pathway and an important pharmacological target.; Chapter 1 of this dissertation provides background information on the biosynthesis of isoprenoids and on the enzymes in the MEP pathway, with a focus on MEP synthase.; The chemical syntheses of the MEP pathway metabolites MEP and 4-diphosphocytidyl-2-C-methyl- D-erythritol (CDPME) are described in Chapter 2. MEP was readily prepared as the sodium salt or as the free acid from commercially available materials with overall yields of 27% (75:25 ex.) and 32% (78:22 ex.), respectively. CDPME was prepared in 40% yield from MEP using a recently developed carbohydrate-nucleotide diphosphate coupling technique.; In a second project described in Chapter 3, recombinant Escherichia coli MEP synthase was purified by chromatography on DE-52 and phenylsepharose, and its steady-state kinetic constants were determined at 37°C and pH = 7.6; kcat = 116 ± s−1, KMDXP = 115 ± 25 μM, and KM NADPH = 0.5 ± 0.2 μM. Synthetic MEP was used to show that the rearrangement/reduction is reversible; Keq37° = 45 ± 6 for DXP and MEP at 150 μM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP; Chapter 4 documents the cloning and identification of a novel squalene synthase from the cyanobacterium Synechocystis sp. The protein lacks a region conserved in homologous eukaryotic proteins that encodes for a membrane-anchoring domain and is naturally soluble. The gene is toxic, however, and methylerythritol is an essential bacterial supplement for transformants. The use of methylerythritol as a supplement for expression of other isoprenoid genes is discussed.
机译：磷酸甲基赤藓糖醇（MEP）途径已被认为是在细菌，藻类和植物叶绿体中产生类异戊二烯化合物的新途径。 MEP合酶，催化1-脱氧- D -木酮糖-5-磷酸酯（DXP）的重排/还原反应，形成2-C-甲基- D -赤藓糖醇- 4-磷酸（MEP），是该途径中的关键步骤，也是重要的药理学靶标。本文的第一章提供了有关类异戊二烯的生物合成和MEP途径中酶的背景信息，重点是MEP合酶。 MEP途径代谢产物MEP和4-diphosphocytidyl-2-C-methyl- D -赤藓糖醇（CDPME）的化学合成在第2章中进行了描述。MEP易于制备为钠盐或从市售材料中获得游离酸，总收率分别为27％（75:25 ex。）和32％（78:22 ex。）。使用最近开发的碳水化合物-核苷酸二磷酸偶联技术，从MEP以40％的收率制备CDPME。在第3章中描述的第二个项目中，重组DEM大肠杆菌MEP合酶通过DE-52和苯基琼脂糖层析纯化，在37°C和pH = 7.6时测定其稳态动力学常数。 k cat = 116±s −1 ，K M DXP = 115±25μM，K M NADPH = 0.5±0.2μM。合成的MEP被用来表明重排/还原是可逆的。 DXP和MEP在150μMNADPH下的K eq 37° = 45±6。使用fosmidomycin和dihydro-NADPH作为末端抑制剂来检查底物结合的机制。二氢-NADPH对NADPH具有竞争模式，对DXP具有非竞争模式。磷霉素是对NADPH的非竞争性抑制剂，其模式代表了对DXP的缓慢，紧密结合的竞争性抑制。这些结果与NADPH在DXP之前结合的有序机制是一致的。第四章从蓝细菌<斜体>囊藻 sp的克隆和鉴定新的角鲨烯合酶。该蛋白质缺少在同源真核蛋白质中保守的区域，该区域编码膜锚定域并且是天然可溶的。但是，该基因具有毒性，而甲基赤藓糖醇是转化子的重要细菌补品。讨论了甲基赤藓糖醇作为其他类异戊二烯基因表达的补充剂的用途。

著录项

  • 作者

    Koppisch, Andrew Thomas.;

  • 作者单位

    The University of Utah.;

  • 授予单位 The University of Utah.;
  • 学科 Chemistry Biochemistry.; Chemistry Organic.
  • 学位 Ph.D.
  • 年度 2002
  • 页码 150 p.
  • 总页数 150
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;有机化学;
  • 关键词

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