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

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.

Paralogous murine Nudt10 and Nudt11 genes have differential expression patterns but encode identical proteins that are physiologically competent diphosphoinositol polyphosphate phosphohydrolases

We previously described paralogous human genes {NUDT10 and NUDT11 [where NUDT is (nucleoside diphosphate attached moiety ‘X’)-type motif, also known as the ‘nudix’-type motif]} encoding type 3 diphosphoinositol polyphosphate phosphohydrolases (DIPP3) [Hidaka, Caffrey, Hua, Zhang, Falck, Nickel, Carrel, Barnes and Shears (2002) J. Biol. Chem. 277, 32730–32738]. Normally, gene duplication is redundant, and lacks biological significance. Is this true for the DIPP3 genes? We address this question by characterizing highly-conserved murine Nudt10 and Nudt11 homologues of the human genes. Thus these genes must have been duplicated prior to the divergence of primates and sciurognath rodents, approx. 115 million years ago, greatly exceeding the 4 million year half-life for inactivation of redundant paralogues; our data therefore indicate that the DIPP3 duplication is unusual in being physiologically significant. One possible functional consequence is gene neofunctionalization, but we exclude that, since Nudt10 and Nudt11 encode identical proteins. Another possibility is gene subfunctionalization, which we studied by conducting the first quantitative expression analysis of these genes. We demonstrated high Nudt10 expression in liver, kidney and testis; Nudt11 expression is primarily restricted to the brain. This differential, but complementary, expression pattern indicates that subfunctionalization is the evolutionary consequence of DIPP3 gene duplication. Our kinetic data argue that diphosphoinositol polyphosphates are more physiologically relevant substrates for DIPP3 than are either diadenosine hexaphosphate or 5-phosphoribosyl 1-pyrophosphate. Thus the significance of the Nudt10/Nudt11 duplication is specific hydrolysis of diphosphoinositol polyphosphates in a tissue-dependent manner.

Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5′,5'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (ApnA, n = 5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)} in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap5A, PP-InsP5, [PP]2-InsP4 and a novel diphosphoinositol polyphosphate ([PP]x-InsPx) in S. pombe using HPLC methods. Ap5A was present at 0.06pmol/mg of protein (approx. 4nM). PP-InsP5, [PP]x-InsPx and [PP]2-InsP4 were present at 15pmol/mg (approx. 1.1μM), 15pmol/mg (approx. 1.1μM) and 30pmol/mg (approx. 2.2μM) respectively, while the intracellular concentration of InsP6 was 0.5nmol/mg of protein (approx. 36μM). Disruption of aps1 resulted in a 52% decrease in Ap6A hydrolase activity in vitro, no detectable change in the intracellular Ap5A concentration, and 3-fold increased intracellular concentrations of PP-InsP5 and [PP]x-InsPx. Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30°C. Overexpression of aps1 via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP]2-InsP4, [PP]x-InsPx or PP-InsP5, but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap5A concentration. Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Ap5A occurred upon overexpression of aps1E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP5 and [PP]x-InsPxin vivo.