Lu Xinyao, Wang Guangsheng, Feng Yue, Liu Song, Zhou Xiaoman, Du Guocheng, Chen Jian
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
J Microbiol Biotechnol. 2016 Oct 28;26(10):1701-1707. doi: 10.4014/jmb.1602.02027.
Lipoxygenase (LOX) is an industrial enzyme with wide applications in food and pharmaceutical industries. The available structure information indicates that eukaryotic LOXs consist of N terminus β-barrel and C terminus catalytic domains. However, the latest crystal structure of LOX shows it is significantly different from those of eukaryotic LOXs, including the N-terminal helix domain. In this paper, the functions of this N-terminal helix domain in the soluble expression and catalysis of LOX were analyzed. Genetic truncation of this helix domain resulted in an insoluble LOX mutant. The active C-terminal domain was obtained by dispase digestion of the LOX derivative containing the genetically introduced dispase recognition sites. This functional C-terminal domain showed raised substrate affinity but reduced catalytic activity and thermostability. Crystal structure analyses demonstrate that the broken polar contacts connecting the two domains and the exposed hydrophobic substrate binding pocket may contribute to the insoluble expression of the C terminus domain and the changes in the enzyme properties. Our data suggest that the N terminus domain of LOX is required for its soluble expression in , which is different from that of the eukaryotic LOXs. Besides this, this N-terminal domain is not necessary for catalysis but shows positive effects on the enzyme properties. The results presented here provide new and valuable information on the functions of the N terminus helix domain of LOX and further improvement of its enzyme properties by molecular modification.
脂氧合酶（LOX）是一种工业酶，在食品和制药行业有广泛应用。现有的结构信息表明，真核脂氧合酶由N端β桶和C端催化结构域组成。然而，最新的脂氧合酶晶体结构显示它与真核脂氧合酶有显著不同，包括N端螺旋结构域。本文分析了该N端螺旋结构域在脂氧合酶可溶性表达和催化中的功能。对该螺旋结构域进行基因截短导致产生了一种不溶性的脂氧合酶突变体。通过对含有基因引入的dispase识别位点的脂氧合酶衍生物进行dispase消化，获得了活性C端结构域。这种功能性C端结构域显示出底物亲和力提高，但催化活性和热稳定性降低。晶体结构分析表明，连接两个结构域的极性接触断裂以及暴露的疏水底物结合口袋可能导致C端结构域的不溶性表达和酶性质的变化。我们的数据表明，脂氧合酶的N端结构域是其在[具体宿主]中可溶性表达所必需的，这与真核脂氧合酶不同。除此之外，该N端结构域对催化不是必需的，但对酶性质有积极影响。本文给出的结果为脂氧合酶N端螺旋结构域的功能以及通过分子修饰进一步改善其酶性质提供了新的有价值信息。
