Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius.
Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius.
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来自嗜热嗜酸革兰氏阳性细菌酸热脂环酸芽孢杆菌的麦芽糖 ATP 结合盒转运蛋白的功能重建。
DOI：
10.1016/j.bbabio.2004.01.005
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
E. Schneider
中科院分区：
文献类型：
--
作者：
F. Scheffel;R. Fleischer;E. Schneider
嗜热嗜酸革兰氏阳性细菌酸热脂环酸芽孢杆菌在60 °C和pH 2-3下生长。该生物体可以利用麦芽糖和麦芽糖糊精作为能量来源，其被ATP结合盒（ABC）输入系统摄取。编码麦芽糖结合蛋白MalE和两个膜整合亚基MalF和MalG的基因聚集在染色体上，但缺乏翻译成同源ATP酶亚基的malK基因。本文报道了以变铅青链霉菌的msiK基因为探针，从基因组DNA中克隆malK基因。纯化后的MalK具有自发的ATP酶活性，Vmax为0.13 μmol Pi/min/mg，Km为330 μM，在该菌生长温度下最适。在大肠杆菌中的malK，malF和malG的共表达导致形成的复合物，可以从亲和基质共洗脱后，膜与dodecylmaltoside溶解。由MalFGK复合物和预先形成的A. acidocaldarius表现出较低的内在ATP酶活性，其被麦芽糖负载的MalE刺激七倍，从而表明ATP水解与底物易位的偶联。这些结果为MalK是A.酸热菌麦芽糖转运蛋白此外，据我们所知，这是第一个报告的功能重建的ABC运输系统从嗜热微生物。
The thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius grows at 60 °C and pH 2–3. The organism can utilize maltose and maltodextrins as energy source that are taken up by an ATP-binding cassette (ABC) import system. Genes encoding a maltose binding protein, MalE, and two membrane-integral subunits, MalF and MalG, are clustered on the chromosome but a malK gene translating into a cognate ATPase subunit is lacking. Here we report the cloning of malK from genomic DNA by using the msiK gene of Streptomyces lividans as a probe. Purified MalK exhibited a spontaneous ATPase activity with a Vmaxof 0.13 μmol Pi/min/mg and a Kmof 330 μM that was optimal at the growth temperature of the organism. Coexpression of malK, malF and malG in Escherichia coli resulted in the formation of a complex that could be coeluted from an affinity matrix after solubilization of membranes with dodecylmaltoside. Proteoliposomes prepared from the MalFGK complex and preformed phospholipid vesicles of A. acidocaldarius displayed a low intrinsic ATPase activity that was stimulated sevenfold by maltose-loaded MalE, thereby indicating coupling of ATP hydrolysis to substrate translocation. These results provide evidence for MalK being the physiological ATPase subunit of the A. acidocaldarius maltose transporter. Moreover, to our knowledge, this is the first report on the functional reconstitution of an ABC transport system from a thermophilic microorganism.
捕获 ATP 结合盒转运蛋白的过渡态：麦芽糖转运协同机制的证据。
DOI：
10.1073/pnas.98.4.1525
发表时间：
2001
影响因子：
11.1
作者：
Chen,J;Sharma,S;Quiocho,FA;Davidson,AL
通讯作者：
Davidson,AL
DOI：
10.1073/pnas.89.6.2360
发表时间：
1992-03-15
影响因子：
11.1
作者：
DAVIDSON, AL;SHUMAN, HA;NIKAIDO, H
通讯作者：
NIKAIDO, H