Cdc14 phosphatases use an intramolecular pseudosubstrate motif to stimulate and regulate catalysis

<p>This dataset contains files of mostly enzymology data on the Cdc14 phosphatases and mutant variants from <em>Saccharomyce cerevisiae </em>and <em>Homo sapiens. </em>It is augmented by structural predictions using AlphaFold2 and some biological experiments with engineered strains of <em>S. cerevisiae </em>and the human opportunistic pathogen <em>Candida albicans </em>looking at effect of <em>CDC14 </em>mutations on cell wall stress sensitivity<em>. </em>The results from these files are reported in a paper of the same title in the Journal of Biological Chemistry. They purpose of the dataset is to provide readers of the JBC paper access to the raw and processed data files used to generate the figures presented in the paper.</p>

<p>Then enzymology data were acquired using several different assays and recombinant enzymes expressed and purified from <em>E. coli</em>. These include steady-state kinetics analyses using the small molecule fluorescent substrate DiFMUP, pre-steady-state kinetics on a stopped flow fluorimeter using DiFMUP, steady-state kinetics using a phosphopeptide substrate and a malachite green-based dye for product detection, and a mass spectrometric assay using a phosphopeptide substrate pool at very low concentration to measure catalytic efficiency as a surrogate for substrate binding. The dataset also include a binding assay using a non-cleavable substrate peptide and peptide trans stimulation assays monitoring activity towards DiFMUP in the presence of various synthetic peptides.</p>

<p>AlphaFold structure predictions were generated using the publicly available AlphaFold2 server and default settings.</p>

<p>The biological experiments consist of standard spot dilution and patch assays comparing growth of yeast strains in the presence and absence of the antifungal drug micafungin.</p>

<p>For context, the abstract of the JBC paper is included below:</p>

<p>Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTP) 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 <em>Saccharomyces cerevisiae</em> Cdc14 we discovered a short sequence in the disordered C-terminus, distal to the catalytic domain, that mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it “<u>s</u>ubstrate-<u>li</u>ke <u>c</u>atalytic <u>e</u>nhancer” (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.</p>