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首页> 外文学位 >Essential fatty acid biosynthetic enzymes of Escherichia coli and Lactococcus lactis subsp. lactis.
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Essential fatty acid biosynthetic enzymes of Escherichia coli and Lactococcus lactis subsp. lactis.

机译：大肠杆菌和乳酸乳球菌亚种的必需脂肪酸生物合成酶。乳酸菌。

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摘要

β-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) encoded by the fabH gene is thought to catalyze the first elongation reaction of type II fatty acid synthesis in bacteria and plant plastids. I report the first bacteria strain that lacking KAS III, a fabH mutant constructed in the gram-positive bacterium Lactococcus lactis subsp. lactis IL1403. Upon supplementation of saturated and unsaturated long-chain fatty acids, a L. lactis Δ fabH strain could be obtained through gene replacement. This fabH mutant strain fails to grow without supplementation with exogenous long-chain fatty acids demonstrating that KASIII plays an essential role in cellular metabolism. The fabH deletion mutant of L. lactis requires only long-chain unsaturated fatty acids for growth, indicating existence of another pathway for saturated fatty acid synthesis. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. I also confirm the essentiality of FabH in Escherichia coli using a plasmid-based gene insertion/deletion system. Together these results provided the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both gram-positive and gram-negative bacteria.; FabG, β-ketoacyl-ACP reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to the β-hydroxyacyl-ACP products. I report the first characterized fabG mutants. By chemical mutagenesis followed by a tritium suicide procedure, I obtained three conditionally-lethal temperature-sensitive fabG (fabGts) mutants. The E. coli mutant has two point mutations: A154T and E233K. In Salmonella enterica Serovar Typhimurium fabGts mutants one strain had a point mutation, S224F whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the two-fold axes of symmetry at the dimer interfaces in this homotetrameric protein suggesting that the quanternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.
机译：由 fabH 基因编码的β-酮酰基-酰基载体蛋白（ACP）合酶III（KAS III）被认为可催化细菌和植物质体中II型脂肪酸合成的首次延伸反应。我报告了第一个缺乏KAS III的细菌菌株，KAS III是在革兰氏阳性细菌乳酸乳球菌亚种中构建的 fabH 突变体。 lactis IL1403。补充饱和和不饱和长链脂肪酸后，<斜体> L。可通过基因置换获得乳酸Δ fabH 菌株。该 fabH 突变菌株在不补充外源长链脂肪酸的情况下无法生长，表明KASIII在细胞代谢中起着重要作用。 乳酸乳杆菌的 fabH 缺失突变体仅需长链不饱和脂肪酸即可生长，表明存在另一种饱和脂肪酸合成途径。实际上，将[1- 14 C]乙酸酯掺入体内脂肪酸表明， fabH 突变体保留了约10％的脂肪酸野生型菌株的合成能力以及该剩余合成能力优先转移至途径的饱和分支。此外，质谱分析表明， fabH 突变体在脂肪酸饥饿时保留了低水平的棕榈酸。我还使用基于质粒的基因插入/缺失系统确认了FabH在大肠杆菌中的必要性。这些结果共同提供了第一个遗传证据，证明FabH在革兰氏阳性和革兰氏阴性细菌中引发II型脂肪酸生物合成的主要缩合反应。 FabG，β-酮酰基-ACP还原酶，可将NADPH依赖性的β-酮酰基-ACP底物还原为β-羟酰基-ACP产物。我报告了第一个特征性的 fabG 突变体。通过化学诱变和a自杀程序，我获得了三个条件致死的温度敏感型 fabG （ fabG ts ）突变体。 E。大肠埃希氏菌突变体有两个点突变：A154T和E233K。在 Salmonella enterica Serovar Typhimurium fabG ts 突变体中，一个菌株具有点突变，即S224F，而第二个菌株则包含两个突变（M125I和A223T）。 FabG突变蛋白的所有已改变残基都位于该同四聚体蛋白的二聚体界面的对称对称轴的两倍或附近。这表明突变FabG蛋白的定量结构可能在非容许温度下被破坏。

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  • 作者

    Lai, Chiou-Yan.;

  • 作者单位

    University of Illinois at Urbana-Champaign.;

  • 授予单位 University of Illinois at Urbana-Champaign.;
  • 学科 Biology Microbiology.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 143 p.
  • 总页数 143
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 微生物学;生物化学;
  • 关键词

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