The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing β-D-mannuronic acid residues (M) into α-L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through β-elimination. The reaction mechanisms for the epimerization and cleavage are similar and some enzymes can perform both. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their activity as well as the amino acids responsible for it are still unknown. In this study, we investigate mechanistic determinants of the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that the lyase activity of AlgE7 is regulated by the type of ion present: Calcium promotes it, whereas NaCl reduces it. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M↓XM and G↓XM, where X can be either M or G. By studying AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 as the catalytic acid.

中文翻译：

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了解双功能 Azotobacter vinelandii mannuronan C-5 差向异构酶和藻酸盐裂解酶 AlgE7 双重活性的机制基础

藻酸盐的功能特性由单体组成和分子量分布决定。甘露糖聚糖 C-5 差向异构酶通过将 β-D-甘露糖醛酸残基 (M) 差向异构化为 α-L-古洛糖醛酸残基 (G) 来确定前者。分子量受海藻酸盐裂解酶的影响，海藻酸盐裂解酶通过β-消除裂解海藻酸盐链。差向异构化和裂解的反应机制相似，一些酶可以同时进行。这些二元酶与没有裂解酶活性的甘露糖醇 C-5 差向异构酶具有高度的序列同一性，其活性背后的机制以及负责它的氨基酸仍然未知。在这项研究中，我们研究了来自Azotobacter vinelandii的 AlgE7 双功能差向异构酶和裂解酶活性的机制决定因素. 基于序列分析，构建了一系列 AlgE7 变体，并通过 NMR 进行活性测定和产品表征。我们的结果表明，AlgE7 的裂解酶活性受存在的离子类型的调节：钙促进它，而 NaCl 减少它。通过使用定义的 poly-M 和 poly-MG 底物，发现 AlgE7 的首选切割位点是 M↓XM 和 G↓XM，其中 X 可以是 M 或 G。通过研究 AlgE7 突变体，R148 被确定为一个重要的裂解酶活性的残基，而点突变体 R148G 导致酶仅具有差向异构酶活性。基于本研究中获得的结果，我们提出了一种统一的差向异构酶和裂合酶催化反应机制，其中 H154 作为催化碱，Y149 作为催化酸。