In vitro biosynthesis of ATP from adenosine and polyphosphate

Chuanqi Sun,Wentian Xu,Zhimin Li,Xiao Ning,Zonglin Li

doi:10.1186/s40643-021-00469-0

Chuanqi Sun, Wentian Xu + Show 3 more

Open Access

https://doi.org/10.1186/s40643-021-00469-0

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Abstract

Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively.Graphical

Highlights

Adenosine triphosphate (ATP) is an energy currency containing high-energy phosphate bonds
ATP plays a key role in signal transduction (Rajendran et al 2016), as it is involved in the synthesis of intracellular second messenger cyclic adenosine monophosphate (Post et al 1998)
adenosine monophosphates (AMP) was phosphorylated into adenosine diphosphates (ADP) and ATP by Polyphosphate kinase (PPK) using p olyP6 as a phosphate donor

Summary

Introduction

Adenosine triphosphate (ATP) is an energy currency containing high-energy phosphate bonds. After screening the suitable PPKs, the temperature, pH, magnesium ion concentration, and enzyme concentration of the coupling reaction were optimized to achieve in vitro production of ATP at different adenosine concentrations. The reactions were conducted in 100 mM Tris–HCl buffer (pH 8.0) at 45 °C containing 10 mM Adenosine, 10 mM ATP, 20 mM MgCl2, appropriate amount of enzymes and different concentrations of polyP6 that ranged from 10 to 50 mM.

Results

Conclusion