Chemoenzymatic synthesis of 3'-phosphoadenosine-5'-phosphosulfate coupling with an ATP regeneration system.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a commonly used anticoagulant drug. Previously, using ATP as the substrate, we had developed a one-pot synthesis to prepare PAPS with 47% ATP conversion efficiency. During the reaction, 47% of ATP was converted into the by-product, ADP. Here, to increase the conversion ratio of ATP to PAPS, an ATP regeneration system was developed to couple with PAPS synthesis. In the ATP regeneration system, the chemical compound, monopotassium phosphoenolpyruvate (PEP- K+ ), was synthesized and used as the phospho-donor. By using 3-bromopyruvic acid as the starting material, the total yield of PEP- K+ synthesis was over 50% at low cost. Then, the enzyme PykA from Escherichia coli was overexpressed, purified, and used to convert the by-product ADP into ATP. When coupled the ATP regeneration system with PAPS synthesis, the higher ratio of PEP- K+ to ADP was associated with higher ATP conversion efficiency. By using the ATP regeneration system, the conversion ratio of ATP to PAPS was increased to 98% as determined by PAMN-HPLC analysis, and 5 g of PAPS was produced in 1 L of the reaction mixture. Furthermore, the chemoenzymatic synthesized PAPS was purified and freeze-dried without observed decomposition. However, the powdery PAPS was more unstable than the PAPS sodium salt in aqueous solution at ambient temperature. This developed chemoenzymatic approach of PAPS production will contribute to the synthesis of heparin, in which PAPS is necessary as the individual sulfo-donor.