An engineered aryl sulfotransferase for the biosynthesis of glycosaminoglycans

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is an essential and expensive sulfonic acid donor, playing a central role in sulfonation modification for glycosaminoglycan biosynthesis. The aryl sulfotransferase (AST) is the primary enzyme responsible for catalyzing the regeneration of PAPS from 3'-phosphoadenosine-5'-phosphate (PAP). However, the application of AST in PAPS regeneration systems has been limited by its poor activity and stability. In this study, we identified a new AST from Hipposideros armiger, named as HaAST. Subsequent protein engineering of HaAST yielded variants exhibiting significantly enhanced catalytic activity and thermal stability. The variant HaAST M13 (D247G/V243E/Y186H/M24I/R187P/R285K/K122N/V25A) exhibiting a 9-fold enhancement in activity and showed 58-fold longer half-life than the reported AST IV at 50 °C. The HaAST M13-catalyzed PAPS regeneration system was successfully employed for the sulfation of glycosaminoglycans, including N- and 2-O-sulfated (NS2S) heparin and chondroitin sulfate (CS), with sulfation degree of 75.31% and 68.68%, respectively. Molecular dynamics (MD) simulations suggested that an enlarged entrance of the substrate pocket, together with increased rigidity in key regions including the substrate channel and binding sites, may contribute to the improvement of both catalytic activity and stability. This study offers an efficient PAPS regeneration system, providing a promising foundation for the enzymatic production of glycosaminoglycans.