A terminal alpha 3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O(2)regulation of the social amoebaDictyosteliumand the parasiteToxoplasma gondii. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, inDictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. InToxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, theToxoplasmaenzyme catalyzes formation of a Gal alpha 1,3Glc alpha linkage rather than the Glc alpha 1,4Glc alpha linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 inToxoplasmaand also in another protist, the crop pathogenPythium ultimum. The fifth sugar is important for glycan function as indicated by the slow-growth phenotype ofgat1 Delta parasites. Computational analyses indicated that, despite the sequence difference, theToxoplasmaglycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases inToxoplasmaandPythiumby an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan inDictyosteliumis a remarkable case of convergent evolution, which emphasizes the importance of the terminal alpha-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

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