Innokentev Aleksei, Schwer Beate, Shuman Stewart
Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, USA.
mBio. 2026 Feb 26:e0394225. doi: 10.1128/mbio.03942-25.
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 Mg-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 LDNC 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.
（i）一种细胞表面酸性磷酸酶Pho1，可从细胞外环境中动员无机磷酸盐；（ii）摄取无机磷酸盐（Pho84、Pho841和Pho842）和甘油磷酸胆碱（Tgp1）的跨膜转运蛋白；以及（iii）分泌型细胞外5'-核苷酸酶（Efn1和Efn2），其可从核糖核苷酸单磷酸（rNMPs）中释放无机磷酸盐，对CMP具有偏好性。芽殖酵母5'-核苷酸酶Sdt1的裂殖酵母同源物SPAC24B11.05的表达在磷酸盐饥饿期间上调，并且该蛋白质积累但不分泌。在此，我们将重组SPAC24B11.05（本文中称为Ifn1，即细胞内5'-核苷酸酶）鉴定为天冬氨酰磷酸酶（HAD）超家族的一种Mg依赖性磷酸水解酶。与对嘧啶单核苷酸和烟酰胺单核苷酸（NMN）具有特异性的Sdt1不同，Ifn1对GMP＞IMP＞CMP＞AMP＞UMP的水解具有偏好性，并且无法水解NMN。Ifn1活性通过特征性LDNC基序中亲核试剂Asp11的丙氨酸突变以及分别预测用于配位核糖羟基和金属辅因子的Asp80和Asp174被丙氨酸替代而被消除。将预测与鸟嘌呤碱基结合的Ifn1 Arg50替换为Sdt1中的相应残基Asn，可增强CMP和AMP的水解，并抑制GMP、IMP和UMP的水解，对NMN则没有活性增加。我们发现催化活性Ifn1的过表达对裂殖酵母有毒性。
重要性
裂殖酵母中的磷酸盐饥饿会触发在磷酸盐动态中具有推定作用的酶的表达增加。许多饥饿诱导的磷酸水解酶被注释为作用于核苷酸，尽管它们的底物特异性尚未被研究。在此，我们将裂殖酵母Ifn1鉴定为天冬氨酰磷酸酶（HAD）超家族中一种饥饿诱导的5'-核苷酸酶，其对GMP和IMP的水解具有偏好性，这使其与同源的芽殖酵母嘧啶特异性5'-核苷酸酶Sdt1区分开来。将Ifn1 Arg50单一替换为Asn（Sdt1中的等效位置）会引发底物转换，表现为对CMP的活性增加以及对GMP和IMP的活性抑制。一个新出现的主题是5'-核苷酸酶的底物特异性是一种可调节的特性。
