Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic.
Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic.
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来自北极的新 PL7 亚家族藻酸盐裂解酶的底物定位机制的结构和分子基础。
DOI：
10.1074/jbc.ra120.015106
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发表时间：
2020
影响因子：
4.8
通讯作者：
Peng Wang
中科院分区：
文献类型：
--
作者：
Fei Xu;Xiu-Lan Chen;Xiao-Hui Sun;Fang Dong;Chun-Yang Li;Ping-Yi Li;Haitao Ding;Yin Chen;Yu-Zhong Zhang;Peng Wang
海藻酸盐裂解酶在海洋海藻酸盐降解过程中起着重要作用。虽然大量的藻酸盐裂解酶已被表征，但对极冷极地环境中的藻酸盐裂解酶知之甚少，这些酶可能具有独特的环境适应和藻酸盐降解机制。在这里，我们报告了一种新的PL7藻酸盐裂解酶AlyC3从冷单胞菌属物种C-3分离的北极棕色海带，包括其系统发育分类，催化性能和结构的表征。我们建议建立一个新的PM特定的亚家族PL7（亚家族6）代表AlyC3的系统发育分析和酶的性质。结构和生化分析表明AlyC3是二聚体，代表了第一个二聚体内藻酸盐裂解酶结构。AlyC3被NaCl激活，并采用一种新的盐激活机制，即盐度通过影响其聚集状态来调节酶活性。我们进一步解析了失活突变体H127A/Y244A与二甘露糖醛酸分子复合的结构，并基于结构和生化分析提出了AlyC3的催化过程。我们发现，Arg82和Tyr190在两端的催化峡谷帮助定位的重复单位的基板和His127，Tyr244，Arg78，和Gln125介导的催化反应。我们的研究首次揭示了海藻酸盐裂解酶中海藻酸盐定位的氨基酸残基，并证明了参与海藻酸盐定位的这些残基在其他海藻酸盐裂解酶中是保守的。本研究为深入了解海藻酸裂解酶降解海藻酸的机理提供了理论依据。
Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties, and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism; that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg82 and Tyr190 at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate and that His127, Tyr244, Arg78, and Gln125 mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase and demonstrates that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases.