Hydrolysis of by-product adenosine diphosphate from 3'-phosphoadenosine-5'-phosphosulfate preparation using Nudix hydrolase NudJ.

摘要: 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of sulfonate group in chemoenzymatic synthesis heparin, a clinically used anticoagulant drug. Previously, we have developed method to synthesize PAPS with Escherichia coli crude extracts, which include three overexpressed enzymes fourth unidentified protein. The unknown protein degrades adenosine diphosphate (ADP), by-product reaction. To further understand control process vitro enzymatic synthesis, decide identify develop defined using purified enzymes. Here, show that Nudix hydrolase NudJ ADP at high efficiency serves as enzyme synthesis. Under condition all 10-mM hydrolyzed form monophosphate (AMP) 15-min better substrate for than triphosphate (ATP). Most importantly, does not cleave product PAPS. removal makes peak more separable from other components chromatographic purification process. This approach production will contribute heparin.