Structure-Guided Engineering of Human 3′-Phosphoadenosine-5′-phosphosulfate Synthetase 1 to Enhance Biosynthesis of PAPS

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the essential sulfate donor for sulfation modifications in crucial biomolecules. Human bifunctional 3'-phosphoadenosine-5'-phosphosulfate synthase 1 (hPAPSS1) directly synthesizes PAPS in two sequential steps, circumventing the inefficiencies of multienzyme systems. However, unsatisfactory ATP substrate affinity and catalytic activity restrict its applications in the biosynthesis of sulfonyl compounds. Herein, a semirational design strategy was employed to enhance its activity, resulting in a positive combinatorial variant, hPAPSS1-C207G/F560W, which exhibited a 2.22-fold increase in activity and achieved a PAPS conversion rate of 34.45%. Molecular dynamics simulations revealed that the C207G substitution enhanced the flexibility of the C-loop, thereby improving its turnover rate. Concurrently, the F560W mutation optimized the conformations of key residues and reduced the binding free energy, enhancing its binding affinity for ATP. This study uncovered two bottleneck issues in enzyme activity and demonstrated mechanisms underlying the hPAPSS1 catalytic performance, enabling efficient synthesis of PAPS.