Mitochondrial ADCK3 Employs an Atypical Protein Kinase-like Fold to Enable Coenzyme Q Biosynthesis

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways. [Display omitted] •A UbiB family protein crystal structure reveals an atypical kinase-like fold•An A-rich loop determines coenzyme selectivity and inhibits autophosphorylation•UbiB-specific features are required for coenzyme Q biosynthesis in vivo•Pathogenic ADCK3 and ADCK4 mutations disrupt protein stability The largely uncharacterized UbiB protein kinase-like family is conserved across all superkingdoms of life. Stefely et al. find that ADCK3, a mitochondrial UbiB protein, adopts an atypical structure with unique features positioned to inhibit protein kinase activity. Altering these features enables in vitro autophosphorylation, but compromises in vivo coenzyme Q biosynthesis.