Protein phosphorylation by eukaryotic protein kinases (ePKs) is a fundamental mechanism of cell signaling in all organisms. In model vertebrates, ~10% of ePKs are classified as pseudokinases, which have amino acid changes within the catalytic machinery of the kinase domain that distinguish them from their canonical kinase counterparts. However, pseudokinases still regulate various signaling pathways, usually doing so in the absence of their own catalytic output. To investigate the prevalence, evolutionary relationships, and biological diversity of these pseudoenzymes, we performed a comprehensive analysis of putative pseudokinase sequences in available eukaryotic, bacterial, and archaeal proteomes. We found that pseudokinases are present across all domains of life, and we classified nearly 30,000 eukaryotic, 1500 bacterial, and 20 archaeal pseudokinase sequences into 86 pseudokinase families, including ~30 families that were previously unknown. We uncovered a rich variety of pseudokinases with notable expansions not only in animals but also in plants, fungi, and bacteria, where pseudokinases have previously received cursory attention. These expansions are accompanied by domain shuffling, which suggests roles for pseudokinases in plant innate immunity, plant-fungal interactions, and bacterial signaling. Mechanistically, the ancestral kinase fold has diverged in many distinct ways through the enrichment of unique sequence motifs to generate new families of pseudokinases in which the kinase domain is repurposed for noncanonical nucleotide binding or to stabilize unique, inactive kinase conformations. We further provide a collection of annotated pseudokinase sequences in the Protein Kinase Ontology (ProKinO) as a new mineable resource for the signaling community.

Glycosyltransferases (GTs) are prevalent across the tree of life and regulate nearly all aspects of cellular functions. The evolutionary basis for their complex and diverse modes of catalytic functions remain enigmatic. Here, based on deep mining of over half million GT-A fold sequences, we define a minimal core component shared among functionally diverse enzymes. We find that variations in the common core and emergence of hypervariable loops extending from the core contributed to GT-A diversity. We provide a phylogenetic framework relating diverse GT-A fold families for the first time and show that inverting and retaining mechanisms emerged multiple times independently during evolution. Using evolutionary information encoded in primary sequences, we trained a machine learning classifier to predict donor specificity with nearly 90% accuracy and deployed it for the annotation of understudied GTs. Our studies provide an evolutionary framework for investigating complex relationships connecting GT-A fold sequence, structure, function and regulation.

1 Glycosyltransferases (GTs) are prevalent across the tree of life and regulate nearly all aspects of 2 cellular functions by catalyzing synthesis of glycosidic linkages between diverse donor and 3 acceptor substrates. Despite the availability of GT sequences from diverse organisms, the 4 evolutionary basis for their complex and diverse modes of catalytic and regulatory functions 5 remain enigmatic. Here, based on deep mining of over half a million GT-A fold sequences from 6 diverse organisms, we define a minimal core component shared among functionally diverse 7 enzymes. We find that variations in the common core and the emergence of hypervariable loops 8 extending from the core contributed to the evolution of catalytic and functional diversity. We 9 provide a phylogenetic framework relating diverse GT-A fold families for the first time and show 10 that inverting and retaining mechanisms emerged multiple times independently during the course 11 of evolution. We identify conserved modes of donor and acceptor recognition in evolutionarily 12 divergent families and pinpoint the sequence and structural features for functional specialization. 13Using the evolutionary information encoded in primary sequences, we trained a machine learning 14 classifier to predict donor specificity with nearly 88% accuracy and deployed it for the annotation 15 of understudied GTs in five model organisms. Our studies provide an evolutionary framework for 16 investigating the complex relationships connecting GT-A fold sequence, structure, function and 17 regulation. 18

Skp1 is a subunit of the SCF (kp1/ullin 1/-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from is hydroxylated by an O-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba , where it regulates SCF assembly and O-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in , generating Galα1, 3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [H]glucose from UDP-[H]glucose to the trisaccharide form of Skp1 in a -dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 inΔ extracts, suggesting specificity for Skp1. -knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.