Substrate recognition mechanism of Streptomyces phospholipase D and enzymatic measurement of plasmalogen
Substrate recognition mechanism of Streptomyces phospholipase D and enzymatic measurement of plasmalogen
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DOI：
10.1016/j.jbiosc.2015.02.020
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发表时间：
2015-10-01
影响因子：
2.8
通讯作者：
Sugimori, Daisuke
中科院分区：
文献类型：
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作者：
Matsumoto, Yusaku;Sugimori, Daisuke
研究了磷脂酶D的底物识别机制和胆碱缩醛磷脂的酶法测定。从链霉菌NA 684菌株的培养上清中纯化磷脂酶D（PLD 684）184倍，回收率为23.7%。在pH 5.0和80 ℃下发现L-α-溶血磷脂酰胆碱（LPC）水解的最大活性。Triton X-100浓度对水解活性有显著影响。在0.05-0.5%和0.1-0.2%（wt/vol）TritonX-100存在下，PLD 684可分别有效地水解1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）和胆碱缩醛磷脂。LPC和胆碱lysoplasmalogen的水解不需要Triton X-100，而是，水解活性被抑制超过0.05%（重量/体积）Triton X-100。该酶对混合胶束底物的选择性强于对脂质体底物的选择性，在1h内可水解98.4%的混合胶束POPC。动力学分析表明，PLD 684水解混合胶束POPC和乳化LPC的限速步骤分别为本体步骤和表面步骤。这些结果表明，PLD 684具有至少两种底物识别机制，以识别具有源自其头部和尾部基团的相当不同的物理性质的各种磷脂。了解PLD 684如何识别底物形式将有助于阐明脂解蛋白在自然界中的作用。此外，我们报告了使用PLD 684和磷脂酶B的胆碱缩醛磷脂酶的酶法测量。这是第一个测定胆碱缩醛磷脂的酶法。(C)2015年，日本生物技术学会。All rights reserved.
The substrate recognition mechanism of phospholipase D and enzymatic measurement of choline plasmalogen were investigated. A phospholipase D (PLD684) from Streptomyces sp. strain NA684 was purified 184-fold from the culture supernatant with 23.7% recovery. Maximum activity for L-alpha-lysophosphatidylcholine (LPC) hydrolysis was found at pH 5.0 and 80 degrees C. The hydrolytic activity was remarkably affected by the concentration of Triton X-100 in the reaction mixture. In the presence of 0.05-0.5% and 0.1-0.2% (wt/vol) Triton X-100, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and choline plasmalogen were efficiently hydrolyzed by PLD684, respectively. Hydrolysis of LPC and choline lysoplasmalogen did not require Triton X-100; rather, the hydrolytic activity was inhibited by more than 0.05% (wt/vol) Triton X-100. The enzyme preferred mixed micelle substrates to liposomal substrates and hydrolyzed 98.4% of mixed micelle POPC in 1 h. Kinetic analysis showed that the rate-limiting steps of hydrolysis of mixed micelle POPC and emulsified LPC by PLD684 were the bulk step and the surface step, respectively. These results suggest that PLD684 has at least two substrate recognition mechanisms to recognize various phospholipids that have considerably different physical properties derived from their head and tail groups. Understanding of how PLD684 recognizes substrate forms will be useful for elucidating roles of lipolytic proteins in nature. Moreover, we report an enzymatic measurement of choline plasmalogen using PLD684 and phospholipase B. This is the first enzymatic method for measuring choline plasmalogen. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.