Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen-specific phospholipase D from Thermocrispum sp.
Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen-specific phospholipase D from Thermocrispum sp.
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DOI：
10.1002/2211-5463.12131
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发表时间：
2016-11
期刊：
影响因子：
2.6
通讯作者：
Sugimori, Daisuke
中科院分区：
文献类型：
--
作者：
Matsumoto, Yusaku;Kashiwabara, Nana;Oyama, Takayuki;Murayama, Kazutaka;Matsumoto, Hideyuki;Sakasegawa, Shin-ich;Sugimori, Daisuke
关键词：
溶血磷脂酶原（LyPls）特异性磷脂酶D（LyPls-PLD）是一种催化LyPls磷酸酯键水解裂解的酶，释放乙醇胺或胆碱和1-（1-烯基）-sn-甘油-3-磷酸（溶胞浆酸）。关于LyPls-PLD和缩醛磷脂（Pls）的代谢途径知之甚少。据报道，人血清/血浆中的Pls水平与几种疾病如阿尔茨海默病和动脉硬化以及各种生物过程（包括细胞凋亡和细胞信号传导）相关。我们从Thermocrispum sp.菌株RD 004668中鉴定了LyPls-PLD，并将该酶纯化、表征、克隆并使用具有His标签的pET 24 a（+）/大肠杆菌表达。该酶的优选底物是胆碱LyPls（LyPlsCho），对乙醇胺LyPls仅具有适度的活性。在最佳条件下（pH 8.0和50 °C），LyPlsCho的稳态动力学分析得出Km和kcat值分别为13.2 μm和70.6 s−1。LyPls-PLD基因的ORF由1005 bp组成，编码334个氨基酸（aa）的蛋白质。LyPls-PLD的氨基酸序列与甘油磷酸二酯磷酸二酯酶（GDPDs）的氨基酸序列高度相似，但底物特异性与GDPDs和一般磷脂酶Ds（PLD）完全不同。结构同源性建模显示LyPls-PLD的两个推定的催化残基（His 46，His 88）与GDPD高度保守。突变和动力学分析表明，LyPls-PLD活性位点的Ala 55、Asn 56和Phe 211可能参与底物识别。这些发现将有助于阐明LyPls-PLD、PLD和GDPD在功能、底物识别机制和生化作用方面的差异。 热卷曲菌属菌株RD 004668及其16 S rDNA序列以NITE BP-01628保藏在NITE专利微生物保藏中心（NPMD;千叶，日本），并以登录号AB 873024保藏在DDBJ数据库中。菌株RD 004668的16 S rDNA和LyPls-PLD基因的核苷酸序列分别以登录号AB 873024和AB 874601保藏在DDBJ数据库中。EC编号EC 3.1.4.4
Lysoplasmalogen (LyPls)‐specific phospholipase D (LyPls‐PLD) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1‐(1‐alkenyl)‐sn‐glycero‐3‐phosphate (lysoplasmenic acid). Little is known about LyPls‐PLD and metabolic pathways of plasmalogen (Pls). Reportedly, Pls levels in human serum/plasma correlate with several diseases such as Alzheimer's disease and arteriosclerosis as well as a variety of biological processes including apoptosis and cell signaling. We identified a LyPls‐PLD from Thermocrispum sp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/Escherichia coli with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions (pH 8.0 and 50 °C), steady‐state kinetic analysis for LyPlsCho yielded K m and k cat values of 13.2 μm and 70.6 s−1, respectively. The ORF of LyPls‐PLD gene consisted of 1005 bp coding a 334‐amino‐acid (aa) protein. The deduced aa sequence of LyPls‐PLD showed high similarity to those of glycerophosphodiester phosphodiesterases (GDPDs); however, the substrate specificity differed completely from those of GDPDs and general phospholipase Ds (PLDs). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls‐PLD were highly conserved to GDPDs. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls‐PLD may participate in the substrate recognition. These findings will help to elucidate differences among LyPls‐PLD, PLD, and GDPD with regard to function, substrate recognition mechanism, and biochemical roles. Thermocrispum sp. strain RD004668 and its 16S rDNA sequence were deposited in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE BP‐01628 and in the DDBJ database under the accession number AB873024. The nucleotide sequences of the 16S rDNA of strain RD004668 and the LyPls‐PLD gene were deposited in the DDBJ database under the accession numbers AB873024 and AB874601, respectively. EC number EC 3.1.4.4
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影响因子：
2.8
作者：
Matsumoto, Yusaku;Sugimori, Daisuke
通讯作者：
Sugimori, Daisuke
影响因子：
5
作者：
Hirano, Yoshitaka;Chonan, Keisuke;Sugimori, Daisuke
通讯作者：
Sugimori, Daisuke
影响因子：
6.7
作者：
Mander, Poonam;Simkhada, Jaya Ram;Yoo, Jin Cheol
通讯作者：
Yoo, Jin Cheol
影响因子：
5.4
作者：
Matsumoto, Yusaku;Mineta, Shingo;Sugimori, Daisuke
通讯作者：
Sugimori, Daisuke
影响因子：
3.4
作者：
Jurkowitz, Marianne S.;Patel, Aalapi;Bell, Charles E.
通讯作者：
Bell, Charles E.