Yamasaki Masayuki, Ogura Kohei, Hashimoto Wataru, Mikami Bunzo, Murata Kousaku
Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
J Mol Biol. 2005 Sep 9;352(1):11-21. doi: 10.1016/j.jmb.2005.06.075.
Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.
海藻酸盐裂解酶通过β-消除反应将海藻酸盐解聚，海藻酸盐是一种由α-L-古洛糖醛酸和β-D-甘露糖醛酸组成的杂多糖。它们的结构/功能关系有望为未来工业海藻酸盐加工以及针对铜绿假单胞菌海藻酸盐生物膜依赖性感染的药物设计提供有价值的信息，但仍有许多未知之处。在此，我们展示了鞘氨醇单胞菌属A1海藻酸盐裂解酶A1-II'的1.0埃分辨率晶体结构及突变分析结果，该酶属于多糖裂解酶（PL）家族7。A1-II'的整体结构采用β-折叠三明治结构，有一个由两个短柔性环覆盖的大活性裂隙。与其他PL-7家族结构比较表明，当催化反应启动时，环打开对于底物结合是必要的。与蛋白质表面许多侧链的无序状态相反，活性裂隙中心的三条相邻β链排列有序。这是由与其他PL-7家族结构相同的氢键网络和堆积样相互作用导致的。通过定点诱变（R146A、E148A、R150A、Q189A和K280A）破坏这些相互作用会使蛋白质不溶或极大降低其活性。A1-II'结构在活性裂隙中包含两个硫酸根离子。硫酸铵是一种强效抑制剂，Ki为2.5 mM，表明我们的结构代表了抑制状态的模型。突变分析结果和连续的氢键网络表明，精氨酸146、谷氨酰胺189、组氨酸191和酪氨酸284形成一个活性中心。酪氨酸284的羟基似乎对催化反应尤为关键，这得到了硫酸根离子结合以及在使用三聚甘露糖醛酸进行能量最小化计算得到的模型中与+1亚位点的C5和O4原子接近程度的支持。本研究所示的结构基础在许多方面与PL-5家族酶相似。
