Schizosaccharomyces pombe adapts to phosphate starvation by upregulating the expression of (i) a cell-surface acid phosphatase, Pho1, that mobilizes inorganic phosphate from the extracellular milieu; (ii) transmembrane transporters that take up inorganic phosphate (Pho84, Pho841, and Pho842) and glycerophosphocholine (Tgp1); and (iii) secreted extracellular 5'-nucleotidase enzymes (Efn1 and Efn2) that release inorganic phosphate from rNMPs, with a preference for CMP. The expression of SPAC24B11.05, a fission yeast homolog of the budding yeast 5'-nucleotidase Sdt1, is upregulated during phosphate starvation, and the protein accumulates without being secreted. Here, we characterized recombinant SPAC24B11.05 (herein Ifn1, for intracellular 5'-nucleotidase) as a Mg2+-dependent phosphohydrolase of the aspartyl-phosphatase (HAD) superfamily. Unlike Sdt1, which is specific for pyrimidine mononucleotides and nicotinamide mononucleotide (NMN), Ifn1 displays a preference for hydrolysis of GMP > IMP > CMP > AMP > UMP and is unable to hydrolyze NMN. Ifn1 activity is abolished by alanine mutations of the Asp11 nucleophile of the signature 11DLDNC15 motif and by alanines in lieu of Asp80 and Asp174 that are predicted to coordinate the ribose hydroxyls and the metal cofactor, respectively. Changing Ifn1 Arg50, which is predicted to engage the guanine nucleobase, to Asn, the corresponding residue in Sdt1, enhances hydrolysis of CMP and AMP and suppresses hydrolysis of GMP, IMP, and UMP, with no gain of activity with NMN. We find that overexpression of catalytically active Ifn1 is toxic to fission yeast.IMPORTANCEPhosphate starvation in fission yeast triggers increased expression of enzymes with imputed roles in phosphate dynamics. Many starvation-induced phosphohydrolases are annotated as acting on nucleotides, though their substrate specificities have not been interrogated. Here, we characterize fission yeast Ifn1 as a starvation-induced 5'-nucleotidase of the aspartyl-phosphatase (HAD) superfamily with a preference for hydrolysis of GMP and IMP that distinguishes it from the homologous budding yeast pyrimidine-specific 5'-nucleotidase Sdt1. A single swap of Ifn1 Arg50 to Asn (the equivalent position in Sdt1) elicits a substrate switch, manifested as a gain of activity with CMP and suppression of activity with GMP and IMP. An emergent theme is that 5'-nucleotidase substrate specificity is a tunable property.
Innokentev et al. (Thu,) studied this question.