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首页> 外文学位 >Enhanced lipogenesis and oxalogenesis in Pseudomonas fluorescens exposed to aluminum stress: A study of intermediary metabolism.
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Enhanced lipogenesis and oxalogenesis in Pseudomonas fluorescens exposed to aluminum stress: A study of intermediary metabolism.

机译：暴露于铝胁迫下的荧光假单胞菌的增脂和草酸生成：中间代谢研究。

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摘要

Pseudomonas fluorescens detoxifies aluminum (Al) in association with oxalic acid and phosphatidylethanolamine (PE). This study was aimed at elucidating the role of the intermediary metabolism of the cell in generating these moieties. Results presented here show a major reconfiguration of the metabolic pathways in response to Al-stress. This metabolic shift allowed the cell to divert precursors towards the production of key metabolites required to synthesize oxalate and PE that are pivotal in Al detoxification. The activity of isocitrate lyase (ICL), an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells while glyoxylate dehydrogenase (GDH), the enzyme mediating the oxidation of glyoxylate to yield oxalate, underwent an 8-fold increase in activity in cells harvested from Al-rich media. There was also a marked increase in activity of such enzymes as pyruvate dehydrogenase (PDH) (4-fold), glucose-6-phosphate dehydrogenase (G6PDH) (1.5-fold), phosphogluconate dehydrogenase (PGDH) (1.5-fold) and a soluble NADP-dependent isocitrate dehydrogenase (IDH) (1.5-fold). However, the activity of alpha-ketoglutarate dehydrogenase (KDH) and a membrane-bound NAD-dependent IDH appeared to diminish by 3- and 1.5-fold respectively. Interestingly, even though the cells were grown in a citrate and/or Al-citrate media, phosphoenolpyruvate carboxykinase (PEPCK) activity appeared to be reduced (1.5-fold) in Al medium. In addition there was no change in activity of malate synthase (MS), an enzyme often expressed in conjunction with isocitrate lyase. The data presented also demonstrate the tendency of these enzymatic activities to return to near control levels once Al has been extruded and immobilized as a gelatinous residue consisting of oxalate and PE. For instance, when transferred to a fresh control medium, Al-stressed cells experienced a sharp decline in ICL activity. Blue native gel electrophoresis and western blot analyses revealed that this fluctuation in ICL activity was modulated by a change in the concentration of the enzyme. 13C NMR analyses revealed the de novo synthesis of PE and oxalate when the cells were exposed to Al. Although the activity of acetyl-CoA carboxylase (ACC) did not show any variation in activity when the cells were subjected to the Al media, the three NADPH generating enzymes, namely G6PDH, PGDH, NADP-dependent IDH did exhibit elevated activities in Al-stressed media. This suggests a deliberate modification of the metabolic pathways that enables the organism to circumvent the stress of Al. The shift in the TCA cycle, glycolysis, the glyoxylate cycle, the pentose phosphate pathway, and gluconeogenesis provides the necessary metabolites to generate the moieties required to immobilize and neutralize the toxic influence of the trivalent metal.
机译：荧光假单胞菌可与草酸和磷脂酰乙醇胺（PE）一起解毒铝（Al）。这项研究旨在阐明细胞中间代谢在产生这些部分中的作用。此处显示的结果显示了响应Al胁迫的代谢途径的重大重构。这种新陈代谢的转变使细胞将前体转移到合成在Al解毒中至关重要的草酸盐和PE所需的关键代谢产物的生产上。异柠檬酸裂合酶（ICL）的活性，参与异柠檬酸裂解成乙醛酸和琥珀酸的酶使铝胁迫的细胞增加4倍，而乙醛酸脱氢酶（GDH）介导乙醛酸的氧化产生草酸，从富含Al的培养基中收获的细胞中的活性增加了8倍。丙酮酸脱氢酶（PDH）（4倍），6磷酸葡萄糖磷酸脱氢酶（G6PDH）（1.5倍），磷酸葡萄糖酸脱氢酶（PGDH）（1.5倍）和α-磷酸酶等酶的活性也显着增加。可溶性NADP依赖性异柠檬酸脱氢酶（IDH）（1.5倍）。但是，α-酮戊二酸脱氢酶（KDH）和与膜结合的NAD依赖性IDH的活性似乎分别降低了3倍和1.5倍。有趣的是，即使细胞在柠檬酸盐和/或柠檬酸铝培养基中生长，磷酸烯醇丙酮酸羧化激酶（PEPCK）活性在铝培养基中似乎也降低了（1.5倍）。此外，苹果酸合酶（MS）的活性没有改变，该酶通常与异柠檬酸裂合酶结合表达。所提供的数据还表明，一旦将Al挤出并固定为由草酸盐和PE组成的凝胶状残基，这些酶活性就会趋于回到接近控制水平。例如，当转移到新鲜的对照培养基中时，铝胁迫细胞的ICL活性急剧下降。蓝色天然凝胶电泳和蛋白质印迹分析表明，ICL活性的这种波动是通过改变酶浓度来调节的。 13C NMR分析显示，当细胞暴露于Al时，PE和草酸盐从头合成。尽管当将细胞置于Al培养基中时，乙酰辅酶A羧化酶（ACC）的活性未显示任何活性变化，但三种NADPH生成酶，即G6PDH，PGDH，NADP依赖性IDH在Al-强调媒体。这表明故意修饰了代谢途径，使生物体能够规避铝的胁迫。 TCA循环，糖酵解，乙醛酸循环，磷酸戊糖途径和糖异生作用的转变提供了必要的代谢产物，以产生固定和中和三价金属的毒性影响所需的部分。

著录项

  • 作者

    Hamel, Robert D.;

  • 作者单位

    University of Waterloo (Canada).;

  • 授予单位 University of Waterloo (Canada).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 231 p.
  • 总页数 231
  • 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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