Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.
Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.
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DOI：
10.1002/mbo3.269
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发表时间：
2015-08
期刊：
影响因子：
3.4
通讯作者：
Yoneyama H
中科院分区：
文献类型：
--
作者：
Kim S;Ihara K;Katsube S;Hori H;Ando T;Isogai E;Yoneyama H
我们先前报道大肠杆菌的alaE基因编码l-丙氨酸输出蛋白AlaE。本研究的目的是阐明AlaE输出的机制。在alaE缺陷的L-丙氨酸非代谢细胞MLA 301 ΔalaE中，L-丙氨酸和L-丙氨酰-L-丙氨酸的最小抑制浓度分别比alaE阳性的亲本细胞MLA 301低4倍和>4000倍，表明AlaE作为外排泵发挥作用，以避免细胞内L-丙氨酸及其衍生物的毒性水平积累。此外，在生理水平的L-丙氨酰-L-丙氨酸存在下，来自L-丙氨酸代谢菌株的alaE缺陷型突变体的生长受到强烈抑制。完整的MLA 301 ΔalaE和MLA 301 ΔalaE/pAlaE细胞产生质粒携带的AlaE，分别积累了MLA 301细胞中检测到的[3 H] l-丙氨酸的约200%和50%，表明AlaE输出l-丙氨酸。将由MLA 301 ΔalaE/pAlaE制备的200 mmol/L L L-丙氨酸反向膜囊泡分别置于200 mmol/L或0.34 μmol/L L L-丙氨酸溶液中，在两种条件下均发生能量依赖性的[3 H]-丙氨酸积累。这种能量依赖性的上坡积累的[3 H] L-丙氨酸强烈抑制在存在下的羰基氰间氯苯腙，但不是由二环己基碳二亚胺，这表明丙氨酸介导的L-丙氨酸挤出驱动的质子动力。基于这些结果，l-丙氨酸输出蛋白的生理作用进行了讨论。
We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [3H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [3H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [3H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.