College of Life Science and Natural Resources, Dong-A University, Busan, 604-714, Korea.
Mol Biol Rep. 2011 Aug;38(6):3741-50. doi: 10.1007/s11033-010-0489-0. Epub 2010 Nov 20.
We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 μmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 μM.
我们使用 RACE PCR 从肉苁蓉愈伤组织中克隆了基因 CdPAL1，使用的是基于其他植物物种已知 PAL 基因的多重序列比对设计的简并引物。该基因与 GenBank 中注册的其他已知 PAL 基因具有高度同源性。重组蛋白表现出 Michaelis-Menten 动力学，Km 为 0.1013 mM，Vmax 为 4.858 μmol min(-1)，Kcat 为 3.36 S(-1)，Kcat/Km 为 33,168 M(-1) S(-1)。当以 L-苯丙氨酸为底物时，该酶的最适 pH 为 8.5，活化能为 38.92 kJ mol(-1)；L-酪氨酸不能作为该蛋白的底物。最适温度为 55°C，热稳定性结果表明，在 70°C 下处理 20 min 后，该蛋白保留了 87%的活性，而在 75°C 下处理 20 min 后，酶活性损失超过 85%。重金属离子（Hg2+、Pb2+和 Zn2+）处理表现出显著的抑制作用。在木质素（肉桂醇、肉桂醛、松柏醛、松柏醇）、苯丙素（肉桂酸、香豆酸、咖啡酸、绿原酸）和苯乙醇（酪醇和红景天苷）生物合成途径的中间体中，只有肉桂酸对 CdPAL1 活性表现出强烈的抑制作用，Ki 为 8 μM。竞争性抑制剂 AIP 表现出很强的抑制作用，Ki=0.056 μM。
