Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli
Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli
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DOI：
10.1074/jbc.275.2.1050
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发表时间：
2000-01-14
影响因子：
4.8
通讯作者：
Bernard, T
中科院分区：
文献类型：
--
作者：
Bourot, S;Sire, O;Bernard, T
外源提供给高渗培养基的渗透保护剂被应激的大肠杆菌细胞有效地导入和积聚，除了在恢复和维持渗透平衡方面具有明显作用外，这些溶质对细胞大分子的行为也应发挥重要作用，例如在蛋白质折叠过程中。采用随机化学诱变方法，获得了条件性赖氨酸营养缺陷型突变体。通过在基本培养基中添加赖氨酸或渗透保护剂（包括甘氨酸甜菜碱（GB）），可以恢复该突变体的生长。生长速率随着细胞内GB浓度的增加成比例地增加。该突变位于 lysA 基因中，导致二氨基庚二酸脱羧酶 (DAPDC) 的 384 位 Ser 被 Phe 取代，从而催化内消旋二氨基庚二酸转化为 L-赖氨酸。我们纯化了野生型 DAPDC 和突变型 DAPDC-sf，并证明 GB 能够在体外激活 DAPDC-sf，从而证实了体内结果。最重要的是，我们表明激活与 DAPDC-sf 的构象变化相关。总而言之，这些结果首次表明，GB 可能以类似伴侣的方式积极协助体内蛋白质折叠。
Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli, In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding, Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.