Identification of two acyl-CoA synthetases from Pseudomonas putida GPo1:: One is located at the surface of polyhydroxyalkanoates granules
Identification of two acyl-CoA synthetases from Pseudomonas putida GPo1:: One is located at the surface of polyhydroxyalkanoates granules
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DOI：
10.1021/bm8001655
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发表时间：
2008-06-01
影响因子：
6.2
通讯作者：
Ren, Qun
中科院分区：
文献类型：
--
作者：
Ruth, Katinka;de Roo, Guy;Ren, Qun
恶臭假单胞菌GPo1能够以细胞内颗粒的形式积累聚羟基烷酸酯（PHA）作为储存材料。分离PHA颗粒并进行蛋白活性分析。从纯化的PHA颗粒中检测到酰基辅酶a合成酶（ACS1）活性。然后从p.p putida GPo1中克隆出相应的基因acs1。利用基因组行走技术，发现并克隆了位于acs1上游的同源acs2。构建了acs1和acs2与编码绿色荧光蛋白（GFP）基因的融合体，并在GPo1中表达。体内荧光显微镜研究表明，ACS1-GFP产生的荧光主要与PHA颗粒有关，而ACS2-GFP产生的荧光主要与细胞膜有关。在对照菌株（仅含GFP）中，荧光在细胞质中均匀分布。我们的结论是，ACS1位于PHA颗粒上，可能在PHA的动员中发挥核心作用，例如，将羟基羧酸单体转化为羟基羧酸辅酶a，这些辅酶a可以进一步被细胞利用。
Pseudomonas putida GPo1 is able to accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules as storage materials. PHA granules were isolated and analyzed for protein activities. An acyl-CoA-synthetase (ACS1) activity was detected from the purified PHA granules. The corresponding gene acs1 was then cloned from P. putida GPo1. With the genomic walking technique, a homologue acs2 located upstream of acs1 was discovered and cloned. Fusions of both acs1 and acs2 with the gene encoding the green fluorescent protein (GFP) were constructed and expressed in GPo1. In vivo fluorescence microscopy studies showed that the fluorescence generated from the ACS1-GFP was mainly associated with the PHA granules, whereas that from ACS2-GFP was mainly with the membrane of the cells. In the control strain (containing GFP alone) fluorescence was distributed evenly in the cytoplasm. We concluded that ACS1 is located on the PHA granules and may play a central role in mobilization of PHA, for example, conversion of hydroxycarboxylic acid monomers to hydroxycarboxyl-CoA, which can be further utilized by the cells.