caa a22 4500 605502730 CHVBK 20210128100607.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6911-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6911-8 Hydrolysis of by-product adenosine diphosphate from 3′-phosphoadenosine-5′-phosphosulfate preparation using Nudix hydrolase NudJ [Elektronische Daten] [Feifei Bao, Huihui Yan, Hanju Sun, Peizhou Yang, Guoqing Liu, Xianxuan Zhou] 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a clinically used anticoagulant drug. Previously, we have developed a method to synthesize PAPS with Escherichia coli crude extracts, which include three overexpressed enzymes and a fourth unidentified protein. The unknown protein degrades adenosine diphosphate (ADP), the by-product of PAPS synthesis reaction. To further understand and control the process of in vitro enzymatic PAPS synthesis, we decide to identify the fourth protein and develop a defined method to synthesize PAPS using purified enzymes. Here, we show that the purified Nudix hydrolase NudJ degrades ADP at high efficiency and serves as the fourth enzyme in PAPS synthesis. Under the defined condition of PAPS synthesis, all of the 10-mM ADP is hydrolyzed to form adenosine monophosphate (AMP) in a 15-min reaction. ADP is a better substrate for NudJ than adenosine triphosphate (ATP). Most importantly, the purified NudJ does not cleave the product PAPS. The removal of ADP makes the PAPS peak more separable from other components in the chromatographic purification process. This developed enzymatic approach of PAPS production will contribute to the chemoenzymatic synthesis of heparin. Springer-Verlag Berlin Heidelberg, 2015 PAPS nationallicence ADP nationallicence Nudix hydrolase nationallicence NudJ nationallicence Heparin nationallicence Bao Feifei School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut Yan Huihui School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut Sun Hanju School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut Yang Peizhou School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut Liu Guoqing School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut Zhou Xianxuan School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10771-10778 0175-7598 99:24<10771 2015 99 253 https://doi.org/10.1007/s00253-015-6911-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6911-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Bao Feifei School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut NATIONALLICENCE 700 1- Yan Huihui School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut NATIONALLICENCE 700 1- Sun Hanju School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut NATIONALLICENCE 700 1- Yang Peizhou School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut NATIONALLICENCE 700 1- Liu Guoqing School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut NATIONALLICENCE 700 1- Zhou Xianxuan School of Biotechnology and Food Engineering, Hefei University of Technology, 230009, Hefei, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10771-10778 0175-7598 99:24<10771 2015 99 253