Loop engineering of aryl sulfotransferase B for improving catalytic performance in regioselective sulfation
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 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 (kcat) and catalytic efficiency (kcat/KM) 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 kcat 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 acitivity of ASTB-OM2 was achieved through molecular docking studies and electron effects of catechol substituents were analyzed by Hammett equation.