In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase
In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase
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DOI：
10.1074/jbc.m115.654798
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发表时间：
2015-07-17
影响因子：
4.8
通讯作者：
Weselake, Randall J.
中科院分区：
文献类型：
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作者：
Pan, Xue;Chen, Guanqun;Weselake, Randall J.
亚麻(Linum usitatisimum L.)许多其他植物含有大量的多不饱和脂肪酸(PUFAs)。卵磷脂(PC)是多不饱和脂肪酸(PUFA)合成的主要部位。这些多不饱和脂肪酸如何从PC进入三酰甘油(TAG)的确切机制需要进一步研究。通过体内和体外方法，我们证明了由酰基辅酶A：溶血磷脂酰基转移酶(LPCAT)的反向作用催化的PC脱酰化反应可以将PC上的多不饱和脂肪酸直接转移到酰基辅酶A池中，使这些多不饱和脂肪酸可用于二酰甘油酰基转移酶(DGAT)催化的反应以产生TAG。分别产生了两种酵母突变体，分别用于体内和体外实验。这两个突变体都提供了零背景，没有内源标签形成能力和极低的LPCAT活性。体内实验表明，在酵母五重突变体中共表达亚麻DGAT1-1和LPCAT1显著增加了TAG中的18碳多不饱和脂肪酸，同时也降低了磷脂中的18碳多不饱和脂肪酸。我们进一步证明，在sn-2-[C-14]酰基-PC孵育后，只有同时含有LPCAT1和DGAT1-1的酵母微体才可能形成[C-14]标签。此外，整个LPCAT1和DGAT1-1偶联过程的比活性显示出更倾向于将C-14标记的亚油酰基或亚麻烯基从PC的sn-2位转移到TAG上。综上所述，我们的数据支持了LPCAT1催化的反向反应和DGAT1-1催化的将多不饱和脂肪酸掺入TAG的反应的生化耦合假说。这一过程代表了在亚麻种子发育过程中丰富多不饱和脂肪酸TAG含量的一条潜在途径。
Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acylCoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[C-14]acyl-PC, formation of [C-14] TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring C-14-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.