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.

中文翻译：

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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。


藻酸盐裂解酶在海洋藻酸盐降解中发挥重要作用。尽管已经对大量藻酸盐裂解酶进行了表征，但人们对极冷极地环境中的藻酸盐裂解酶知之甚少，这些裂解酶可能具有独特的环境适应和藻酸盐降解机制。在这里，我们报告了来自 Psychromonas sp. 的新型 PL7 海藻酸裂解酶 AlyC3 的表征。从北极褐藻海带中分离出的 C-3，包括其系统发育分类、催化特性和结构。基于系统发育分析和酶学特性，我们建议建立以 AlyC3 为代表的新的 PM 特异性 PL7 亚家族（亚家族 6）。结构和生化分析表明，AlyC3 是二聚体，代表第一个二聚体内切藻酸盐裂解酶结构。 AlyC3由NaCl激活，采用新型盐激活机制；也就是说，盐度通过影响其聚集状态来调整酶活性。我们进一步解析了与二甘露糖醛酸分子复合的失活突变体 H127A/Y244A 的结构，并基于结构和生化分析提出了 AlyC3 的催化过程。我们发现催化峡谷两端的 Arg82 和 Tyr190 有助于底物重复单元的定位，His127、Tyr244、Arg78 和 Gln125 介导催化反应。我们的研究首次揭示了藻酸盐裂解酶中藻酸盐定位的氨基酸残基，并证明参与藻酸盐定位的此类残基在其他藻酸盐裂解酶中是保守的。这项研究使人们更好地了解藻酸盐裂解酶降解藻酸盐的机制。