Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Plant J. 2018 Oct;96(2):287-299. doi: 10.1111/tpj.14029. Epub 2018 Aug 24.
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and committed step in the Kennedy pathway for triacylglycerol (TAG) biosynthesis and, as such, elucidating its mode of regulation is critical to understand the fundamental aspects of carbon metabolism in oleaginous crops. In this study, purified Brassica napus diacylglycerol acyltransferase 1 (BnaDGAT1) in n-dodecyl-β-d-maltopyranoside micelles was lipidated to form mixed micelles and subjected to detailed biochemical analysis. The degree of mixed micelle fluidity appeared to influence acyltransferase activity. BnaDGAT1 exhibited a sigmoidal response and eventual substrate inhibition with respect to increasing concentrations of oleoyl-CoA. Phosphatidate (PA) was identified as a feed-forward activator of BnaDGAT1, enabling the final enzyme in the Kennedy pathway to adjust to the incoming flow of carbon leading to TAG. In the presence of PA, the oleoyl-CoA saturation plot became more hyperbolic and desensitized to substrate inhibition indicating that PA facilitates the transition of the enzyme into the more active state. PA may also relieve possible autoinhibition of BnaDGAT1 brought about by the N-terminal regulatory domain, which was shown to interact with PA. Indeed, PA is a key effector modulating lipid homeostasis, in addition to its well recognized role in lipid signaling. BnaDGAT1 was also shown to be a substrate of the sucrose non-fermenting-1-related kinase 1 (SnRK1), which catalyzed phosphorylation of the enzyme and converted it to a less active form. Thus, this known regulator of carbon metabolism directly influences TAG biosynthesis.
二酰基甘油酰基转移酶 1（DGAT1）催化三酰基甘油（TAG）生物合成的肯尼迪途径中的最后一个和关键步骤，因此，阐明其调节方式对于理解油脂作物碳代谢的基本方面至关重要。在这项研究中，在正十二烷基-β-D-麦芽糖苷胶束中纯化的油菜二酰基甘油酰基转移酶 1（BnaDGAT1）被脂质化为形成混合胶束，并进行了详细的生化分析。混合胶束的流动性似乎影响酰基转移酶活性。BnaDGAT1 对增加的油酰辅酶 A 浓度表现出 S 形响应和最终的底物抑制。磷酸酯（PA）被鉴定为 BnaDGAT1 的前馈激活剂，使 Kennedy 途径中的最后一种酶能够适应导致 TAG 的碳流入。在 PA 的存在下，油酰辅酶 A 的饱和度图变得更加双曲线并且对底物抑制不敏感，表明 PA 促进了酶向更活跃状态的转变。PA 还可能缓解由 N 端调节域引起的 BnaDGAT1 可能的自动抑制，该调节域与 PA 相互作用。事实上，PA 是除了其在脂质信号传导中的公认作用之外，还调节脂质稳态的关键效应物。BnaDGAT1 也被证明是蔗糖非发酵-1 相关激酶 1（SnRK1）的底物，该酶催化酶的磷酸化并将其转化为活性较低的形式。因此，这种已知的碳代谢调节剂直接影响 TAG 生物合成。
