Pandey Brijesh, Pandey Veda Prakash, Dwivedi Upendra Nath
Department of Biochemistry, University of Lucknow, Lucknow 226007, Uttar Pradesh, India.
Protein Expr Purif. 2011 Oct;79(2):197-203. doi: 10.1016/j.pep.2011.06.003. Epub 2011 Jun 25.
A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.
从银合欢的次生木质部中克隆出一个编码肉桂醇脱氢酶（CAD）的cDNA，该酶催化肉桂醛转化为相应的肉桂醇。克隆的cDNA在大肠杆菌BL21(DE3)pLysS细胞中表达。温度和锌离子对该酶的表达和活性起着关键作用，在18°C和2 mM锌离子条件下，CAD以活性酶的形式在可溶部分中最大程度地表达。表达的蛋白质在镍-氮三乙酸琼脂糖柱上纯化了14.78倍达到同质，比活性为346 nkat/mg蛋白质。纯化后的酶对肉桂醇（12.2 μM）的米氏常数最低，其次是松柏醇（18.1 μM）和芥子醇（23.8 μM）。该酶对肉桂醇（超过20 μM）和松柏醇（超过100 μM）表现出高底物抑制作用。对CAD蛋白的计算机分析显示出四个特征性共有序列，分别为GHEXXGXXXXXGXXV；C(100)、C(103)、C(106)、C(114)；GXGXXG以及C(47)、S(49)、H(69)、L(95)、C(163)、I(300)，它们分别参与催化锌离子结合、结构锌离子结合、烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）结合和底物结合。使用Modeller 9v5生成的三级结构呈现出一个具有凸起的结构锌离子结合结构域的三叶结构。催化锌离子结合结构域、底物结合结构域和NADP(+)结合结构域形成了一个由两个主要叶保护的口袋。酶催化、序列同源性和三维模型均支持克隆的CAD属于植物醇脱氢酶家族。
