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Inositol phosphate signaling in plants is of substantial agricultural interest, with a considerable focus on the inositol tris/tetrakisphosphate kinase (ITPK) family of inositol phosphate kinases. Historically, the 4–6 isoforms of ITPKs that higher plants each express have been studied for their multiplexing a metabolic pathway to synthesize inositol hexakisphosphate (ie InsP6 or phytate), through the phosphorylation and dephosphorylation of multiple inositol phosphates, including Ins(1,3,4,5,6)P5 (inositol‐1,3,4,5,6‐pentakisphosphate). A more recent discovery is ITPK‐catalyzed phosphorylation of InsP6 to inositol pyrophosphates, which regulate plant immunity and phosphate homeostasis. However, a molecular‐based explanation for these alternate catalytic activities has been missing, because no plant ITPK structure has previously been solved. Herein, we provide biochemical and structural analyses of ITPKs from Zea mays and Glycine max. For this work we introduce a simple, enzyme‐coupled microplate‐based assay of InsP6 kinase activity that should promote more general access to this important field. Furthermore, a ZmITPK1/InsP6 crystal complex is described at a resolution of 2.6 Å, which identifies a number of catalytically important residues; their functionality is confirmed by mutagenesis. We further demonstrate that ZmITPK1 adds a β‐phosphate to the 3‐position of Ins(1,2,3,4,5)P5, yielding a candidate signal for regulating phosphate homeostasis. An impactful discovery is our description of a 29‐residue catalytic specificity element; by interchanging this element between GmITPK1 and GmITPK2, we demonstrate how its isoform‐specific sequence specifically determines whether the host protein phosphorylates InsP6, without substantially affecting Ins(1,3,4,5,6)P5 metabolism. Our structural rationalization of key catalytic differences between alternate ITPK isoforms will complement future research into their functional diversity. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP 5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP 5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Technical challenges have to date prevented a complete profiling of the levels of myo -inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP 5 and 2-PP-InsP 5 . The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP 5 and 2-PP-InsP 5 . In summary, our study substantially expands PP-InsP biology in mammals., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)