Asp1 from Schizosaccharomyces pombe Binds a [2Fe-2S]²⁺ Cluster Which Inhibits Inositol Pyrophosphate 1-Phosphatase Activity

Abstract

Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron–sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1371–920), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1371–920 contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe–S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S]2+ cluster in Asp1371–920 was demonstrated by UV–visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S]2+ cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S]2+ cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity.

Resonance Raman spectroscopy of Fe–S proteins and their redox properties

“…Being sensitive to the type, ligands and configuration of a cluster, RR spectroscopy has been playing an important part in these discoveries. Some of the recent achievements include the evidence for the presence of a [2Fe–2S] 2+ cluster in a kinase/phosphatase Asp1 that regulates cell morphogenesis in yeasts [9], characterization of the [4Fe–4S] 2+ cluster in HydF, a protein involved in the maturation of organometallic H cluster of Fe–Fe hydrogenase [4] and elucidation of the missing pieces (i.e., the transient catalytic intermediates) of the catalytic cycle puzzle in hydrogenases [7, 82], which provide the key information about biological hydrogen activation. We believe that RR spectroscopy has a bright future in illuminating the structure and function of Fe–S clusters that are still to come to our lab benches.…”

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

“…Preparation of Recombinant Proteins-Recombinant human DIPP1 and the PPIP5K2 kinase domain were prepared as described previously (17,39). We also constructed pDEST515 plasmids (45) hosting wild-type hPPIP5K1 (BC057395.1) and hPPIP5K2 (NM_001345875), each with an N-terminal FLAG tag.…”

“…2). The latter enzymes are of general interest; in addition to hosting a kinase domain that phosphorylates 5-InsP 7 to InsP 8 , PPIP5Ks posses a separate phosphatase domain that dephosphorylates InsP 8 back to 5-InsP 7 (15)(16)(17).…”

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures

“…Two highly conserved classes of enzymes can generate IPPs: the IP6K family and the Vip1 (PPIP5K) family. Vip1 family members are bifunctional enzymes consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain, which in vitro specifically hydrolyze the IPPs generated by the Vip1 kinase domain (196,199).…”