Abstract：
Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTPs) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14, we discovered a short sequence in the disordered C terminus, distal to the catalytic domain, which mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it \"substrate-like catalytic enhancer\" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.
摘要：
Cdc14磷酸酶在结构和机械上与蛋白质酪氨酸磷酸酶（PTP）相关，但对主要由细胞周期蛋白依赖性激酶沉积的磷酸Ser-Pro-X-Lys/Arg位点进化出独特的特异性。这种专业化在真核生物中广泛保守。Cdc14活性位点的进化重新配置以选择性地适应phoSer-Pro可能需要对规范的PTP催化循环进行修饰。在研究酿酒酵母Cdc14时，我们在无序的C端发现了一个短序列，催化域的远端，模拟最佳底物。动力学分析表明，这种假底物结合活性位点并强烈刺激限速磷酸酶水解，我们将其命名为“类似底物的催化增强剂”(SLiCE)。SLiCE基序在所有Dikarya真菌Cdc14直向同源物中都有发现，并且含有不变的谷氨酰胺，我们建议通过类似基材的接触来定位，以帮助水解水的取向，类似于Cdc14缺乏的其他PTP中的保守活性位点谷氨酰胺。AlphaFold2预测显示，脊椎动物Cdc14直向同源物包含与活性位点结合的保守的C末端α螺旋。尽管显然与真菌序列无关，该基序还使底物状接触，并在催化袋中具有不变的谷氨酰胺。改变人Cdc14A和Cdc14B中的这些残基表明其通过与真菌基序相同的机制起作用。然而,真菌和脊椎动物SLiCE基序在功能上不可互换，阐明催化过程中潜在的活性位点差异。最后,我们证明真菌SLiCE基序是Cdc14活性的磷酸化调控靶标。我们的研究揭示了Cdc14磷酸酶中异常刺激的假底物基序的进化。
