The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365−920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371−920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe–S cluster in Asp1365−920 inside the cell. However, we show that the Fe–S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe–S cluster in vivo that is not involved in its pyrophosphatase activity.

Aggregation-Induced Emission of Au/Ag Alloy Nanoclusters for Fluorescence Detection of Inorganic Pyrophosphate and Pyrophosphatase Activity

The development of sensitive and accurate detection of inorganic pyrophosphate (PPi) and pyrophosphatase activity (PPase) is important as they play vital roles in biological systems. However, it is still not satisfactory for most of the analytical methods for PPi and PPase because of their Cu2+-dependence and poor accuracy. Although the metal ion triggered aggregation-induced emission (AIE) of metal nanoclusters (NCs) offers a new approach to design a Cu2+-free strategy for the accurate determination of PPi and PPase recently, current methods are all focused on utilizing pure metal NCs. Alloy NCs incorporating the advantages of diverse metal usually can achieve improved behaviors in the application, such as enhanced sensitivity and stability. In this work, glutathione stabilized alloy Au/Ag NCs were synthesized via a simple method and used for the fluorescence detection of PPi and PPase based on a Zn2+-regulated AIE strategy. The controlled release of Zn2+ by PPi and PPase could regulate the AIE of Au/Ag NCs and be employed to response PPi concentration and PPase activity. This method processes simple procedure, high sensitivity and stability, and low toxicity. In addition, we also studied the AIE behaviors of this Au/Ag NCs and offer some fundamental understanding of the AIE properties of water-soluble alloy NCs. This study not only provides a straightforward and new approach for PPi and PPase determination but a basis for further study on the AIE properties of alloy NCs and their application.

Monitoring inorganic pyrophosphatase activity with the fluorescent dizinc(ii) complex of a macrocycle bearing one dansylamidoethyl antenna

The dizinc(ii) complex of a hexaazamacrocycle with an antenna allowed monitoring of the PPi hydrolysis by using inorganic pyrophosphatase in real-time.

Quantum Dot Doping-Induced Photoluminescence for Facile, Label-Free, and Sensitive Pyrophosphatase Activity Assay and Inhibitor Screening

Development of simple, convenient, and sensitive assay methods for pyrophosphatase (PPase) activity is of importance, for disease diagnosis and drug discovery. Herein, a simple, rapid, label-free, and sensitive fluorescence sensor for PPase activity assay is developed, using Cu2+ doping-induced quantum dot (QD) photoluminescence as a signal reporter. The Cu2+ doping of ZnSe QD can induce a dopant-dependent emission response, which will be inhibited after the premixing of Cu2+ with pyrophosphate (PPi), to form a Cu2+-PPi complex. Then, the hydrolysis of PPi into phosphate (Pi), specifically catalyzed by PPase, liberates the free Cu2+ to regain the QD doping for the fluorescence response, which is highly dependent on the PPase activity. The PPase can be sensitively and selectively assayed, with a detection limit of 0.1 mU/mL. The developed sensing strategy can be also employed for the PPase inhibitor screening. Thus, the current QD doping-based sensing strategy offers an efficient and promising avenue for Cu2+, PPi, or PPase-related target analysis, and might hold great potential for the further applications in the clinical disease diagnosis.