Structural and biochemical characterisation of a novel alginate lyase from Paenibacillus sp. str. FPU-7
Structural and biochemical characterisation of a novel alginate lyase from Paenibacillus sp. str. FPU-7
复制标题
DOI：
10.1038/s41598-019-51006-1
复制
发表时间：
2019-10-16
影响因子：
4.6
通讯作者：
Kimoto, Hisashi
中科院分区：
文献类型：
--
作者：
Itoh, Takafumi;Nakagawa, Emi;Kimoto, Hisashi
一种新的海藻酸裂解酶，PsAly，分子量为33 kDa，其氨基酸序列与其他已知的蛋白质没有显着的相似性，从类芽孢杆菌属菌株FPU-7的生化和结构特征。PsAly的最大活性在65 ℃下获得，最适pH为pH 7-7.5。二价阳离子，如Mg 2+，Mn 2+，或Co 2+，和金属螯合剂，乙二胺四乙酸抑制的活性增强。反应产物表明PsAly是一种内溶酶，对聚甘露糖醛酸具有偏好。在这里，我们报告了PsAly的详细晶体结构，分辨率为0.89埃，它具有一个β-螺旋折叠，产生一个长裂缝。该酶的催化位点与其它多糖裂解酶不同。保守残基的定点突变分析预测Tyr 184和Lys 221为催化残基，分别从C5质子中提取并向糖苷键提供质子。发现一种阳离子结合到裂缝的底部并中和底物的羧基，降低C5质子的pK（a）以促进催化。我们的研究提供了一个深入了解的结构基础的催化海藻酸裂解酶和β-螺旋多糖裂解酶。
A novel alginate lyase, PsAly, with a molecular mass of 33 kDa and whose amino acid sequence shares no significant similarity to other known proteins, was biochemically and structurally characterised from Paenibacillus sp. str. FPU-7. The maximum PsAly activity was obtained at 65 degrees C, with an optimum pH of pH 7-7.5. The activity was enhanced by divalent cations, such as Mg2+, Mn2+, or Co2+, and inhibited by a metal chelator, ethylenediaminetetraacetic acid. The reaction products indicated that PsAly is an endolytic enzyme with a preference for polymannuronate. Herein, we report a detailed crystal structure of PsAly at a resolution of 0.89 angstrom, which possesses a beta-helix fold that creates a long cleft. The catalytic site was different from that of other polysaccharide lyases. Site-directed mutational analysis of conserved residues predicted Tyr184 and Lys221 as catalytic residues, abstracting from the C5 proton and providing a proton to the glycoside bond, respectively. One cation was found to bind to the bottom of the cleft and neutralise the carboxy group of the substrate, decreasing the pK(a) of the C5 proton to promote catalysis. Our study provides an insight into the structural basis for the catalysis of alginate lyases and beta-helix polysaccharide lyases.