新疆紫草羧基-辅酶A连接酶(AeCCLs)的全长克隆及功能验证

Cloning and functional verification of carboxyl CoA ligases(AeCCLs) in Arnebia euchroma

羧基辅酶A连接酶(carboxyl CoA ligases, CCLs)是腺苷酸合成酶基因家族的一个重要分支,主要存在2步催化反应,在三磷酸腺苷的存在下能够可逆的催化不同结构的羧酸盐焦磷酸化形成相应的酰基-单磷酸腺苷中间体;辅酶A或其他酰基受体的硫醇基中的自由电子通过亲核攻击取代单磷酸腺苷形成相应的硫酯。该文以新疆紫草Arnebia euchroma作为研究材料,基于前期转录组数据,筛选到2条AeCCLs基因,命名为AeCCL5(XP019237476.1)和AeCCL7(XP019237476.1),生物信息学分析推测相对分子质量分别为60.569 kDa和60.928 kDa,理论等电点(theoretical pI)分别为8.59和8.92,不稳定系数(instability index)分别为36.78和39.27,存在跨膜结构域,不存在信号肽。通过多序列比对及进化树分析发现,AeCCLs与其他植物的同源蛋白序列相似性不高,这类酶的底物结合位点不是高度保守的,可能是在进化过程中,序列和结构可以适应新底物的变化而变化。该研究首次将AeCCL5和AeCCL7基因全长克隆到表达载体pCDFDuet-1上,原核表达带有His标签的AeCCL5和AeCCCL7蛋白,并通过镍柱纯化得到AeCCL5和AeCCL7的纯化蛋白,通过体外酶促反应证明了AeCCL5和AeCCL7均可参与紫草素生物合成的上游苯丙烷代谢途径,催化4-香豆酸生成4-香豆酰-辅酶A,进而合成紫草素类化合物生物合成的重要前体之一——对羟基苯甲酸。

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP019237476.1) and AeCCL7(XP019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.