title: Hydrolysis of by-product adenosine diphosphate from 3'-phosphoadenosine-5'-phosphosulfate preparation using Nudix hydrolase NudJ.
abstract: 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.
authors: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China.|Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus, Xuancheng, 242000, China.
authors: Feifei
authors: F
authors: Bao
authors: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China.|Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus, Xuancheng, 242000, China.
authors: Huihui
authors: H
authors: Yan
authors: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China.|Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus, Xuancheng, 242000, China.
authors: Hanju
authors: H
authors: Sun
authors: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China.|Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus, Xuancheng, 242000, China.
authors: Peizhou
authors: P
authors: Yang
authors: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China.|Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus, Xuancheng, 242000, China.
authors: Guoqing
authors: G
authors: Liu
authors: School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China. zhoux@hfut.edu.cn.|Wanjiang Institute of Poultry Technology, Hefei University of Technology, Xuancheng Campus, Xuancheng, 242000, China. zhoux@hfut.edu.cn.
authors: Xianxuan
authors: X
authors: Zhou
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D006868:Hydrolysis; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism*; D000096964:Nudix Hydrolases
chemical_list: D000244:Adenosine Diphosphate; D010724:Phosphoadenosine Phosphosulfate; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases
keywords: ADP; Heparin; NudJ; Nudix hydrolase; PAPS