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首页> 外文OA文献 >Pathways of glycine betaine synthesis in two extremely halophilic bacteria, Actinopolyspora halophila and Ectothiorhodospira halochloris
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Pathways of glycine betaine synthesis in two extremely halophilic bacteria, Actinopolyspora halophila and Ectothiorhodospira halochloris

机译：两种极端嗜盐细菌，嗜盐放线菌和嗜盐球藻嗜硫菌中甘氨酸甜菜碱合成的途径

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摘要

Glycine betaine is a solute which is able to restore and maintain the osmotic balance of living cells. In this thesis, the glycine betaine synthesis in two extremely halophilic bacteria Actinopolyspora halophila and Ectothiorhodospira halochloris is investigated.A. halophila synthesized remarkably high intracellular concentrations of glycine betaine. The highest glycine betaine concentration, determined at 24% (w/v) NaCl, was 33% of the cellular dry weight.The data presented in this work indicate that the de novo synthesis of glycine betaine proceeds via the threefold methylation of glycine. S-adenosylmethionine acts as the methyl group donor in the reactions. The genes encoding this pathway were cloned and successfully expressed in Escherichia coli. In E. halochloris, glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) catalyze the reaction sequence. In A. halophila all three methylation reactions appear to be catalyzed by a fusion protein. The methyltransferases from the two bacteria show high sequence homology.Furthermore, it was demonstrated that in addition to the glycine methylation pathway, A. halophila has the ability to oxidize choline to glycine betaine. Choline was first oxidized to betaine aldehyde in a reaction in which H2O2-generation and oxygen consumption are coupled. Betaine aldehyde was oxidized further to glycine betaine in a reaction in which NAD(P)+ was reduced.The GSMT and SDMT of E. halochloris were expressed in E. coli, purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificity and pH optima near physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent Km and Vmax values were determined for all substrates, when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Glycine betaine inhibited the methylation reactions only at high concentrations.Finally, it was demonstrated that the expression of the E. halochloris methyltransferase genes in E. coli results in glycine betaine accumulation and improves salt tolerance.
机译：甘氨酸甜菜碱是一种溶质，能够恢复和维持活细胞的渗透平衡。本文研究了两种极端嗜盐细菌嗜盐放线菌和嗜盐球藻嗜硫菌中甘氨酸甜菜碱的合成。嗜盐菌合成了细胞内高浓度的甘氨酸甜菜碱。最高甘氨酸甜菜碱浓度为24％（w / v）NaCl，为细胞干重的33％。这项工作中提供的数据表明，甘氨酸甜菜碱的从头合成是通过甘氨酸的三倍甲基化进行的。 S-腺苷甲硫氨酸在反应中充当甲基供体。克隆了编码该途径的基因，并在大肠杆菌中成功表达。在盐生大肠杆菌中，甘氨酸肌氨酸N-甲基转移酶（GSMT）和肌氨酸二甲基甘氨酸N-甲基转移酶（SDMT）催化反应顺序。在嗜盐曲霉中，所有三个甲基化反应似乎都由融合蛋白催化。两种细菌的​​甲基转移酶具有高度的序列同源性。此外，还证明除嗜盐菌甲基化途径外，嗜盐曲霉还具有将胆碱氧化为甘氨酸甜菜碱的能力。胆碱首先在反应中被氧化成甜菜碱醛，其中生成H2O2和消耗氧气。在还原NAD（P）+的反应中，甜菜碱醛被进一步氧化为甘氨酸甜菜碱。盐毒大肠杆菌的GSMT和SDMT在大肠杆菌中表达，纯化，并表征了它们的一些酶学性质。两种酶在生理pH附近都具有高底物特异性和最适pH。没有发现辅助因子的证据。酶显示出其底物的米氏（Michaelis-Menten）动力学。当另一种底物以饱和浓度存在时，确定所有底物的表观Km和Vmax值。两种酶均被反应产物S-腺苷同型半胱氨酸强烈抑制。甘氨酸甜菜碱仅在高浓度时抑制甲基化反应。最后，证明了大肠杆菌中卤化大肠杆菌的甲基转移酶基因的表达导致甘氨酸甜菜碱的积累并提高了耐盐性。

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    Nyyssölä Antti;

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  • 年度 2001
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