Abstract

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP.

Key Points

• PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP.

• PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides.

• PPK2c forms polyphosphate granules in vitro from any NTP.

中文翻译：

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一种通用的多磷酸激酶：真养产碱杆菌的 PPK2c 接受嘌呤和嘧啶核苷酸，包括尿苷二磷酸。

摘要

多磷酸激酶 (PPK) 催化三磷酸核苷的 γ-磷酸基团可逆转移至不断增长的多磷酸链。大多数已知的 PPK 是 ATP 特异性的，但有些也可以使用 GTP 作为磷酸盐供体。在这项研究中，我们描述了 β-变形菌Ralstonia eutropica的 PPK2 型 PPK 的特性。纯化的酶 (PPK2c) 高度非特异性，接受嘌呤核苷酸以及吡啶核苷酸（包括 UTP）作为底物。PolyP 引物的存在对于活性来说不是必需的。相应的核苷二磷酸和显微镜可检测的聚磷酸盐颗粒被鉴定为反应产物。如果聚磷作为磷酸盐供体存在，PPK2c 还会催化相应的二磷酸核苷形成 ATP、GTP、CTP、dTTP 和 UTP。值得注意的是，还检测到了大量的四磷酸核苷 AT(4)P、GT(4)P、CT(4)P、dTT(4)P 和 UT(4)P。PPK2c 的低核苷酸特异性注定了该酶与 PolyP 结合成为生物技术应用中 ATP 和其他核苷酸再生的强大工具。例如，PPK2c 和 polyP 用于替代 ATP，并仅用催化量的 ADP 为己糖激酶催化的葡萄糖磷酸化提供燃料。

关键点

•富养罗尔斯克氏菌的PPK2c 可用于任何NTP 或dNTP 的再生。

• PPK2c 高度非特异性，接受所有嘌呤和嘧啶核苷酸。

• PPK2c在体外由任何 NTP形成聚磷酸盐颗粒。