Closed-Loop System Driven by ADP Phosphorylation from Pyrophosphate Affords Equimolar Transformation of ATP to 3′-Phosphoadenosine-5′-phosphosulfate
Closed-Loop System Driven by ADP Phosphorylation from Pyrophosphate Affords Equimolar Transformation of ATP to 3′-Phosphoadenosine-5′-phosphosulfate
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DOI：
10.1021/acscatal.1c02004
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发表时间：
2021-08
期刊：
影响因子：
12.9
通讯作者：
Ruirui Xu;Yang Wang;Hao Huang;Xuerong Jin;Jianghua Li;G. Du;Zhen Kang
中科院分区：
文献类型：
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作者：
Ruirui Xu;Yang Wang;Hao Huang;Xuerong Jin;Jianghua Li;G. Du;Zhen Kang
3′-磷酸腺苷-5 ′-磷酸硫酸酯（3′-Phosphoadenosine-5′-phosphosulfate，PAPS）是生物体中所有硫酸化反应的通用硫酸基团供体。生物制造含硫酸盐化合物如肝素和硫酸软骨素的雄心也促进了体外合成中对PAP的研究。然而，已建立的PAPS的酶促合成面临天然低的理论转化率（50%（两个ATP到一个PAPS））和高成本的障碍。在这里，我们开发了一种PAPS合成路线，理论转化率为100%，提供等摩尔的ATP转化为PAPS。通过融合已鉴定的不同物种的腺苷5′-三磷酸硫酸化酶和腺苷5′-磷酸硫酸激酶，我们创造了一种人工活性双功能酶，直接将ATP转化为PAPS。为了最大化从ATP到PAPS的转化，通过筛选消耗低成本聚P作为磷酸供体的聚P激酶来设计和工程化聚磷酸盐（polyP）依赖性ATP再生系统。此外，我们还发现PPi可以作为磷酸供体，通过polyP激酶将ADP磷酸化为ATP。在证明聚P激酶中PPi激酶活性的广泛分布后，理论上创建了闭环ATP再生途径以将一个ATP转化为一个PAPS。以PPi为磷酸供体，ATP转化为PAPS的转化率达92.3%。本研究所建立的PAPS低成本高效酶法合成路线，将促进硫酸化化合物和多肽的生物合成。
3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is a universal sulfate group donor for all biological sulfation reactions in living organisms. Ambitions to biomanufacture sulfate-containing compounds such as heparin and chondroitin sulfate also promote the study on PAPSin vitrosynthesis. However, the established enzymatic synthesis of PAPS faces hurdles of the natural low theoretical transformation rate of 50% (two ATP to one PAPS) and high cost. Here, we developed a PAPS synthesis route with 100% theoretical transformation rate which affords equimolar transformation of ATP to PAPS. By fusing the identified adenosine 5′-triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase from different species, we created an artificial active bifunctional enzyme to directly convert ATP to PAPS. To maximize the conversion from ATP to PAPS, a polyphosphate (polyP)-dependent ATP regeneration system was designed and engineered by screening polyP kinases which consumes the low-cost polyP as the phosphate donor. In addition, we found that PPi could be used as the phosphate donor for phosphorylating ADP to ATP by polyP kinases. After demonstration of the wide distribution of PPi kinase activity in polyP kinases, a closed-loop ATP regeneration route was thereupon created to convert one ATP to one PAPS in theory. Using PPi as the phosphate donor, the conversion rate of ATP to PAPS reached 92.3%. The efficient enzymatic route that is constructed here for PAPS synthesis with low cost would boost the biosynthesis of sulfated compounds and peptides.