Closed-Loop System Driven by ADP Phosphorylation from Pyrophosphate Affords Equimolar Transformation of ATP to 3′-Phosphoadenosine-5′-phosphosulfate

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is a universal sulfate group donor for all biological sulfation reactions in living organisms. Ambitions to biomanufacture sulfate-containing compounds such as heparin and chondroitin sulfate also promote the study on PAPS in vitro synthesis. However, the established enzymatic synthesis of PAPS faces hurdles of the natural low theoretical transformation rate of 50% (two ATP to one PAPS) and high cost. Here, we developed a PAPS synthesis route with 100% theoretical transformation rate which affords equimolar transformation of ATP to PAPS. By fusing the identified adenosine 5'-triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase from different species, we created an artificial active bifunctional enzyme to directly convert ATP to PAPS. To maximize the conversion from ATP to PAPS, a polyphosphate (polyP)-dependent ATP regeneration system was designed and engineered by screening polyP kinases which consumes the low-cost polyP as the phosphate donor. In addition, we found that PPi could be used as the phosphate donor for phosphorylating ADP to ATP by polyP kinases. After demonstration of the wide distribution of PPi kinase activity in polyP kinases, a closed-loop ATP regeneration route was thereupon created to convert one ATP to one PAPS in theory. Using PPi as the phosphate donor, the conversion rate of ATP to PAPS reached 92.3%. The efficient enzymatic route that is constructed here for PAPS synthesis with low cost would boost the biosynthesis of sulfated compounds and peptides.

Automated summary

The article introduces a new method for PAPS synthesis with a theoretical conversion rate of 100%, which directly converts ATP into PAPS by designing a new enzymatic route, achieving high efficiency and low cost.