Obeid, H. | bioRxiv Science

bioRxiv2026

Exploring the substrate specificity and catalytic mechanism in a PAPS independent ASST from Salmonella typhimurium

Sulfation plays an important role in many metabolic processes such as substrate detoxification, solubilization or activation. The enzymes responsible for these reactions, sulfotransferases, occur in all domains of life and can be classified in two major, evolutionary independent groups, PAPS dependent and PAPS independent or arysulfate sulfotransferases (ASST) enzymes. Besides the fact that they play key roles in biological functions, sulfotransferases can be interesting enzymatic tools to selectively sulfate specific positions of various metabolites. To this end we need to understand the relation between sequence, structure and function. Actually, eukaryotic PAPS dependent sulfotransferases have extensively been studied, while we largely lack knowledge about the diversity of bacterial PAPS-independent ASSTs. We have conducted a multidisciplinary study of a bacterial sulfotransferase from Salmonella typhimurium, revealing the importance of active site residues for activity and guiding substrate specificity. Based on the crystal structure in complex with substrate, we used molecular docking combined with Quantum Mechanics/Molecular Mechanics (QM/MM) free energy calculations to investigate the reaction catalytic mechanism with a focus on the role of specific active site residues in substrate specificity. Our results show that the catalytic mechanism involving three conserved histidines is common with previously determined ASSTs, and highlight a specific pair of aromatic residues, Phe174 and Tyr557, that play an important role in defining the substrate specificity in ASST clade 5. These findings enhance our general understanding of how substrate specificity is attained and has the potential to guide further enzyme engineering to expand the acceptor scope of ASSTs.

biochemistry↗

bioRxiv2026

Mechanistic Basis of Sulfo Transfer in a PAPS-Independent ArylSulfate SulfoTransferases Revealed by Crystallography and QM/MM Simulations

Enzymatic sulfation offers a green-chemistry route to the selective sulfation of phenolic and other hydroxylated compounds. Bacterial arylsulfate sulfotransferases (ASSTs) are particularly attractive biocatalysts because they use simple aryl sulfate donors, such as p-nitrophenyl sulfate (pNPS), instead of the complex and labile cofactor PAPS required by conventional sulfotransferases. However, their rational application and engineering remain limited because the molecular principles controlling substrate recognition, catalytically competent donor and acceptor positioning, covalent sulfo-enzyme formation, and sulfo transfer are not fully understood. In particular, it has remained unclear how conserved active-site histidines cooperate during catalysis and how active-site preorganization promotes efficient sulfo transfer. Here, we address these questions for the newly characterized ASST (ASTB) from Desulfitobacterium hafniense clade 6 (Dh_ASST_6) by combining X-ray crystallography, steady-state enzyme kinetics, and quantum mechanics/molecular mechanics (QM/MM) simulations. Crystal structures of Dh_ASST_6 in the apo state and donor-soaked state reveal a monomeric {beta}-propeller enzyme with a histidine-centered active site and directly identify His350 as the transient sulfo carrier. Kinetic analysis shows efficient turnover and supports a ping-pong bi-bi mechanism, consistent with formation of a covalent sulfo-enzyme intermediate. Steered QM/MM simulations of the second catalytic step show that sulfuryl transfer from sulfated His350 to the phenolic acceptor proceeds through a proton-coupled, multistep pathway in which His283 assists acceptor activation by proton transfer. Active-site preorganization precedes the main bond-reorganization event and generates a near-attack geometry in which acceptor alignment, proton transfer, and stabilization of the sulfo group by a conserved hydrogen-bonding network jointly promote sulfo transfer. Free-energy reconstruction gives a low barrier of approximately 9.6 kcal/ mol, in agreement with efficient enzymatic turnover. The structural, kinetic, and computational data support a complete catalytic cycle for Dh_ASST_6 based on a cooperative two-histidine mechanism. This model establishes catalytically competent acceptor positioning and preservation of the histidine-centered hydrogen-bonding network as key design principles for ASST engineering. These features provide concrete targets for expanding substrate scope and tuning donor or acceptor preference while maintaining efficient sulfuryl transfer.

biochemistry↗