Repeated Evolution of Inactive Pseudonucleases in a Fungal Branch of the Dis3/RNase II Family of Nucleases

The RNase II family of 3′–5′ exoribonucleases is present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like “pseudonucleases.” The latter function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. However, the evolutionary origins of these pseudonucleases are unknown: What sequence of events led to their novel function, and when did these events occur? Here, we show how RNase II pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. In contrast, N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the nonnuclease function requires these regions. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid “titan” growth stages. This phenotype of C. neoformans Ssd1/Dis3L2 deletion is consistent with those of inactive fungal pseudonucleases, yet the protein retains an active site sequence signature. We propose that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, whereas the RNase activity was lost repeatedly in independent lineages.     

The N-Terminal GTPase Domain of p190RhoGAP Proteins Is a PseudoGTPase

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The double lives of phosphatases of regenerating liver: A structural view of their catalytic and noncatalytic activities

Phosphatases of regenerating liver (PRLs) are protein phosphatases involved in the control of cell growth and migration. They are known to promote cancer metastasis but, despite over 20 years of study, there is still no consensus about their mechanism of action. Recent work has revealed that PRLs lead double lives, acting both as catalytically active enzymes and as pseudophosphatases. The three known PRLs belong to the large family of cysteine phosphatases that form a phosphocysteine intermediate during catalysis. Uniquely to PRLs, this intermediate is stable, with a lifetime measured in hours. As a consequence, PRLs have very little phosphatase activity. Independently, PRLs also act as pseudophosphatases by binding CNNM membrane proteins to regulate magnesium homeostasis. In this function, an aspartic acid from CNNM inserts into the phosphatase catalytic site of PRLs, mimicking a substrate–enzyme interaction. The delineation of PRL pseudophosphatase and phosphatase activities in vivo was impossible until the recent identification of PRL mutants defective in one activity or the other. These mutants showed that CNNM binding was sufficient for PRL oncogenicity in one model of metastasis, but left unresolved its role in other contexts. As the presence of phosphocysteine prevents CNNM binding and CNNM-binding blocks catalytic activity, these two activities are inherently linked. Additional studies are needed to untangle the intertwined catalytic and noncatalytic functions of PRLs. Here, we review the current understanding of the structure and biophysical properties of PRL phosphatases.     

Trypanosomatid Deoxyhypusine Synthase Activity Is Dependent on Shared Active-Site Complementation between Pseudoenzyme Paralogs

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FAM105A/OTULINL Is a Pseudodeubiquitinase of the OTU-Class that Localizes to the ER Membrane

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Human pantothenate kinase 4 is a pseudo‐pantothenate kinase

Pantothenate kinase generates 4′‐phosphopantothenate in the first and rate‐determining step of coenzyme A (CoA) biosynthesis. The human genome encodes three well‐characterized and nearly identical pantothenate kinases (PANK1‐3) plus a putative bifunctional protein (PANK4) with a predicted amino‐terminal pantothenate kinase domain fused to a carboxy‐terminal phosphatase domain. Structural and phylogenetic analyses show that all active, characterized PANKs contain the key catalytic residues Glu138 and Arg207 (HsPANK3 numbering). However, all amniote PANK4s, including human PANK4, encode Glu138Val and Arg207Trp substitutions which are predicted to inactivate kinase activity. Biochemical analysis corroborates bioinformatic predictions—human PANK4 lacks pantothenate kinase activity. Introducing Glu138Val and Arg207Trp substitutions to the human PANK3 and plant PANK4 abolished their robust pantothenate kinase activity. Introducing both catalytic residues back into human PANK4 restored kinase activity, but only to a low level. This result suggests that epistatic changes to the rest of the protein already reduced the kinase activity prior to mutation of the catalytic residues in the course of evolution. The PANK4 from frog, an anamniote living relative encoding the catalytically active residues, had only a low level of kinase activity, supporting the view that HsPANK4 had reduced kinase activity prior to the catalytic residue substitutions in amniotes. Together, our data show that human PANK4 is a pseudo‐pantothenate kinase—a catalytically deficient variant of the catalytically active PANK4 found in plants and fungi. The Glu138Val and Arg207Trp substitutions in amniotes (HsPANK3 numbering) completely deactivated the pantothenate kinase activity that had already been reduced by prior epistatic mutations.