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. 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. Loop engineering of aryl sulfotransferase B improves catalytic performance in regioselective sulfation.