Caldo Kristian Mark P, Greer Michael S, Chen Guanqun, Lemieux M Joanne, Weselake Randall J
Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada.
Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
FEBS Lett. 2015 Mar 12;589(6):773-8. doi: 10.1016/j.febslet.2015.02.008. Epub 2015 Feb 14.
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in the acyl-CoA-dependent triacylglycerol biosynthesis. Although the first DGAT1 gene was identified many years ago and the encoded enzyme catalyzes a key step in lipid biosynthesis, no detailed structure-function information is available on the enzyme due to difficulties associated with its purification. This study describes the purification of recombinant Brassica napus DGAT1 (BnaC.DGAT1.a) in active form through solubilization in n-dodecyl-β-D-maltopyranoside, cobalt affinity chromatography, and size-exclusion chromatography. Different BnaC.DGAT1.a oligomers in detergent micelles were resolved during the size-exclusion process. BnaC.DGAT1.a was purified 126-fold over the solubilized fraction and exhibited a specific activity of 26 nmol TAG/min/mg protein. The purified enzyme exhibited substrate preference for α-linolenoyl-CoA>oleoyl-CoA=palmitoyl-CoA>linoleoyl-CoA>stearoyl-CoA.
二酰基甘油酰基转移酶1（DGAT1）催化依赖酰基辅酶A的三酰甘油生物合成的最后一步。尽管多年前就已鉴定出首个DGAT1基因，且该基因编码的酶催化脂质生物合成中的关键步骤，但由于其纯化存在困难，目前尚无关于该酶的详细结构-功能信息。本研究描述了通过在正十二烷基-β-D-麦芽糖苷中溶解、钴亲和层析和尺寸排阻层析，以活性形式纯化重组甘蓝型油菜DGAT1（BnaC.DGAT1.a）的方法。在尺寸排阻过程中，分离出了去污剂胶束中的不同BnaC.DGAT1.a寡聚体。BnaC.DGAT1.a比溶解部分纯化了126倍，比活性为26 nmol TAG/分钟/毫克蛋白。纯化后的酶对α-亚麻酸酰基辅酶A>油酰基辅酶A=棕榈酰基辅酶A>亚油酰基辅酶A>硬脂酰基辅酶A表现出底物偏好性。
