Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping

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.

Cleavage-Polyadenylation Factor Cft1 and SPX Domain Proteins Are Agents of Inositol Pyrophosphate Toxicosis in Fission Yeast

“…13A ). Note that Aps1-type IPP pyrophosphatase enzymes are also active as endopolyphosphatases ( 38 , 39 ), raising the possibility that the increase in polyphosphate in aps1 Δ cells reflects a contribution of Aps1 to polyphosphate turnover. The salient finding here is that the increase in polyphosphate content in the spx1 Δ strain is eliminated in asp1 Δ spx1 Δ and asp1-D333A spx1 Δ double mutants, where the polyphosphate levels mirror those of the asp1 Δ and asp1-D333A single mutants ( Fig.…”

Translational approach to increase phosphate accumulation in two plant species through perturbance of inositol pyrophosphates

“…While data from earlier literature suggested that 1-InsP 7 rather than 5-InsP 7 is the ideal substrate for DDP1 (Lonetti et al ., 2011), a more recent study shows that DDP1 can also hydrolyze 5-InsP 7 , and preferentially hydrolyzes the 5-position PP bond from 1,5-InsP 8 rather than from the 1-position in vitro (Kilari et al ., 2013). A very recent and extensive crystallography study provides a molecular explanation for this in that PP-InsPs have several binding modes in the DDP1 catalytic pocket, with some being more productive for hydrolysis than others (Márquez-Moñino et al ., 2021). This work established that 1-InsP 7 binds in a more productive mode, whereas 5-InsP 7 molecules bind less productively within DDP1.…”

“…Although DDP1 can also act on other substrate signaling molecules in yeast and animals, there is currently no evidence to suggest that these other potential substrates are present in plants. These alternative substrates include two distinct types of molecules: polyphosphates (polyP; polyP n ) and diadenosine polyphosphates (Ap n As; specifically, Ap 5 A and Ap 6 A), which are known substrates for DDP1 in S. cerevisiae (Safrany et al ., 1999; Lonetti et al ., 2011; Andreeva et al ., 2019; Márquez-Moñino et al ., 2021). Both molecular classes have also been linked to maintaining P i metabolism and cellular homeostasis in a variety of prokaryotic and eukaryotic organisms (Lorenzo-Orts et al ., 2020; Pietrowska-Borek et al ., 2020).…”

“…To achieve this, we used a yeast phosphatase to artificially remove plant PP-InsPs. We selected d iadenosine and d iphosphoinositol p olyphosphate phosphohydrolase (DDP1; gene YOR163w), a phosphatase from Saccharomyces cerevisiae containing a NUDIX hydrolase motif that hydrolyzes phosphoanhydride bonds from InsP 7 and InsP 8 , polyphosphates (polyP), and diadenosine polyphosphates (Ap n A; specifically, Ap 5 A and Ap 6 A) (Safrany et al ., 1999; Lonetti et al ., 2011; Kilari et al ., 2013; Márquez-Moñino et al ., 2021). Of these three molecule classes, only PP-InsPs have been detected in plants (Desai et al ., 2014; Laha et al ., 2015) and have a known synthesis pathway (Desai et al ., 2014; Laha et al ., 2015; Adepoju et al ., 2019; Laha et al ., 2019).…”

Supramolecular interaction of inositol phosphates with Cu(ii): comparative study of InsP6–InsP3

“…Fig.12Role of H bonding to coordinated water molecules in Mg(II) complexes. Top: 6WOD (disordered 6WOE is analogous, both structures contain the same Homo sapiens hydrolase); 56 bottom: 7AUU 57. Octahedral coordination mode is almost unchanged from structure to structure (yeast hydrolase).…”