Abstract
The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.
Highlights
Inositol polyphosphates (InsPs) are small molecules, primarily derived from myo-inositol, the phosphorylation patterns of which generate second messengers, essential metabolites, or cofactors involved in a variety of cell processes [1]
Our ScDDP1:PCP-InsP7 complex was obtained at acidic pH at which the enzyme is not active (Fig. 2H); and due to residue conservation, we suggest that fully active ScDDP1 will show three Mg2+
We crystallized DDP1 in the presence of HMP; the density we found in the active site is in agreement with a polyphosphate chain of at least 15 units
Summary
Inositol polyphosphates (InsPs) are small molecules, primarily derived from myo-inositol, the phosphorylation patterns of which generate second messengers, essential metabolites, or cofactors involved in a variety of cell processes [1]. Three different families of phosphatases that are able to hydrolyze PP-InsPs at least in vitro have been identified, belonging in terms of their fold to the Nudix hydrolase [human diphosphoinositol polyphosphate phosphohydrolases (DIPPs)], tyrosine phosphatase (ScSiw, essential for prion propagation), or histidine acid phosphatase (Vip enzymes) families. Regarding the latter, Vip and Asp yeast kinases are bifunctional enzymes containing tethered kinase and phosphatase domains
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