Purification and characterization of 1-acyl-sn-glycerol-3-phosphate acyltransferase with a substrate preference for polyunsaturated fatty acyl donors from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10
Purification and characterization of 1-acyl-sn-glycerol-3-phosphate acyltransferase with a substrate preference for polyunsaturated fatty acyl donors from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10
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DOI：
10.1093/jb/mvy025
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发表时间：
2018-07-01
影响因子：
2.7
通讯作者：
Kurihara, Tatsuo
中科院分区：
文献类型：
--
作者：
Ogawa, Takuya;Tanaka, Asako;Kurihara, Tatsuo
1-酰基-sn-甘油-3-磷酸酰基转移酶（在细菌中称为PlsC）催化溶血磷脂酸的酰化，并负责磷脂酸的从头产生，磷脂酸是合成各种膜甘油磷脂的前体。由于PlsC是一种完整的膜蛋白，因此通常难以在不引起其失活的情况下将其溶解，尽管其普遍存在和生理重要性，但这一直阻碍其生化表征。大多数PlsC的生物化学研究已经进行了使用粗膜制剂或完整的细胞。在这项研究中，我们成功地溶解和纯化的重组PlsC在其活性形式从二十碳五烯酸生产细菌希瓦氏菌利文斯顿Ac 10使用6-环己基-1-己基-β-D-麦芽糖苷作为洗涤剂。我们的特点是纯化的酶，并发现它有一个多不饱和脂肪酰基，如二十碳五烯酰基的酰基供体的底物偏好。这些结果为纯化PlsC家族酶提供了一种新的方法，并证明了具有独特底物特异性的新PlsC的存在。
1-Acyl-sn-glycerol-3-phosphate acyltransferase (designated as PlsC in bacteria) catalyzes the acylation of lysophosphatidic acid and is responsible for the de novo production of phosphatidic acid, a precursor for the synthesis of various membrane glycerophospholipids. Because PlsC is an integral membrane protein, it is generally difficult to solubilize it without causing its inactivation, which has been hampering its biochemical characterization despite its ubiquitous presence and physiological importance. Most biochemical studies of PlsC have been carried out using crude membrane preparations or intact cells. In this study, we succeeded in solubilization and purification of a recombinant PlsC in its active form from the eicosapentaenoic acid-producing bacterium Shewanella livingstonensis Ac10 using 6-cyclohexyl-1-hexyl-beta-D-maltoside as the detergent. We characterized the purified enzyme and found that it has a substrate preference for the acyl donors with a polyunsaturated fatty acyl group, such as eicosapentaenoyl group. These results provide a new method for purification of the PlsC family enzyme and demonstrate the occurrence of a new PlsC with unique substrate specificity.