Activities and structure-function analysis of fission yeast inositol pyrophosphate (IPP) kinase-pyrophosphatase Asp1 and its impact on regulation of pho1 gene expression Journal Article
| Authors: | Benjamin, B.; Garg, A.; Jork, N.; Jessen, H. J.; Schwer, B.; Shuman, S. |
| Article Title: | Activities and structure-function analysis of fission yeast inositol pyrophosphate (IPP) kinase-pyrophosphatase Asp1 and its impact on regulation of pho1 gene expression |
| Abstract: | Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1–385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP g-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1’s kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro by virtue of its antagonism of IP turnover. © 2022 American Society for Microbiology. All rights reserved. |
| Keywords: | controlled study; unclassified drug; genetics; nonhuman; protein domain; metabolism; gene; gene expression; carboxy terminal sequence; in vitro study; enzyme activity; recombinant enzyme; structure activity relation; mutational analysis; phosphorylation; gene expression regulation; alanine; adenosine triphosphate; indoles; enzyme specificity; indole derivative; phosphate; protein kinase; phosphotransferase; hydrolysis; synthesis; schizosaccharomyces; schizosaccharomyces pombe proteins; enzyme active site; schizosaccharomyces pombe protein; fission yeast; fungal enzyme; regulon; biochemical analysis; pyrophosphatases; inositol phosphate; pyrophosphate; diphosphates; phosphotransferases (phosphate group acceptor); article; inositol phosphates; inositol polyphosphate kinase; inorganic pyrophosphatase; inositol pyrophosphates; keywords asp1; 1 inositol pyrophosphate 7; 1,5 inositol pyrophosphate 8; 5 inositol pyrophosphate 7; adenosine triphosphate gamma phosphate; asp1 protein; inositol derivative; inositol pyrophosphate 6; inositol pyrophosphate 6 kinase; inositol pyrophosphate 8; inositol pyrophosphate kinase; asp1 protein, s pombe; diphosphoric acid; indolepropanol phosphate; multifunctional enzyme; pho1 gene; multifunctional enzymes |
| Journal Title: | mBio |
| Volume: | 13 |
| Issue: | 3 |
| ISSN: | 2150-7511 |
| Publisher: | American Society for Microbiology |
| Date Published: | 2022-06-01 |
| Start Page: | e01034-22 |
| Language: | English |
| DOI: | 10.1128/mbio.01034-22 |
| PUBMED: | 35536002 |
| PROVIDER: | scopus |
| PMCID: | PMC9239264 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 August 2022 -- Source: Scopus |
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