Catechol sulfates act in our body as important antioxidants and often have anti-inflammatory properties. Enzymatic sulfation of catechol as well as substituted catechols by aryl sulfotransferases opens up an environmentally-friendly route for chemo- and/or regioselective sulfation. This study focuses on reengineering of loop12 and loop13 of aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense in order to improve the sulfate transfer efficiency of six catechols. The obtained ASTB variants were generated in a KnowVolution campaign using the random mutagenesis method SeSaM and the multi-site saturation method OmniChange. The catalytic activity (k_cat) and catalytic efficiency (k_cat/K_M) of the final variant ASTB-OM2 (Q191Y/Y218W/L225V) were improved for all six investigated catechols when compared to ASTB-WT (e.g., 13.6-fold improvement of k_cat for 3-bromocatechol). HPLC-MS analysis confirmed the improved sulfate stoichiometry of ASTB-OM2 with a transfer efficiency up to 94% for 3-methylcatechol in comparison to 24% for ASTB-WT. A molecular understanding of the improved sulfation activity of ASTB-OM2 was achieved through molecular docking studies and electron effects of catechol substituents were analyzed by Hammett equation.

中文翻译：

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芳基磺基转移酶B的环工程以提高区域选择性硫酸化的催化性能

硫酸邻苯二酚在人体中起重要的抗氧化剂的作用，通常具有抗炎作用。芳基磺基转移酶将邻苯二酚和取代的邻苯二酚进行酶促硫酸化，为化学和/或区域选择性硫酸化开辟了一条环境友好的途径。这项研究侧重于改造来自哈芬脱硫杆菌的芳基磺基转移酶B（ASTB）的loop12和loop13，以提高六个邻苯二酚的硫酸盐转移效率。使用随机诱变方法SeSaM和多位点饱和方法OmniChange在KnowVolution活动中生成获得的ASTB变体。催化活性（k _cat）和催化效率（k _cat / K _M与ASTB-WT相比，最终变体ASTB-OM2（Q191Y / Y218W / L225V）的所有六种邻苯二酚均得到改善（例如，3-溴邻苯二酚的k _cat改善了13.6倍）。HPLC-MS分析证实，ASTB-OM2的硫酸盐化学计量得到了改进，3-甲基邻苯二酚的转移效率高达ASTB-WT的24％，转移效率高达94％。通过分子对接研究获得了对ASTB-OM2硫酸盐活性提高的分子理解，并通过Hammett方程分析了邻苯二酚取代基的电子效应。