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Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP
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  • Kaifang Liu,
  • Xiulai Chen,
  • Yunlu Zhong,
  • Jia Liu,
  • Guipeng Hu,
  • Cong Gao,
  • Liang Guo,
  • Wei Song,
  • Liming Liu
Kaifang Liu
Jiangnan University
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Xiulai Chen
Jiangnan University
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Yunlu Zhong
Jiangnan University
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Jia Liu
Jiangnan University
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Guipeng Hu
Jiangnan University
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Cong Gao
Jiangnan University
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Liang Guo
Jiangnan University
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Wei Song
Jiangnan University
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Liming Liu
Jiangnan Univ.
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Abstract

The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.