Dataset Information

Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

ABSTRACT: The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize and quantify InsPs in complex samples is limited. Here, we have synthesized and applied symmetrically and unsymmetrically ¹³C-labeled myo-inositol and inositol phosphates. These probes were utilized in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to investigate InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR-down to individual enantiomers-which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme contributing to cellular homeostasis. Detailed characterization of MINPP1 activity in vitro and in cells showcased the unique reactivity of this phosphatase. Our results demonstrate that metabolic labeling with stable isotopomers in conjunction with NMR spectroscopy and CE-MS constitutes a powerful tool to annotate InsP networks in a variety of biological contexts.

SUBMITTER: Nguyen Trung M

PROVIDER: S-EPMC9801504 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

Nguyen Trung Minh M Kieninger Stefanie S Fandi Zeinab Z Qiu Danye D Liu Guizhen G Mehendale Neelay K NK Saiardi Adolfo A Jessen Henning H Keller Bettina B Fiedler Dorothea D

ACS central science 20221205 12

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize and quantify InsPs in complex samples is limited. Here, we have synthesized and applied symmetrically and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes ...[more]

PMID: 36589890

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Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

Dataset Information

Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

Publications

Stable Isotopomers of <i>myo-</i>Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

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