Crystal structure of a fungal dihydroxyacetone kinase reveals a non-canonical ATP-binding site.

Abstract

Dihydroxyacetone kinase (DAK) catalyzes the ATP-dependent phosphorylation of dihydroxyacetone (DHA) and is an important enzyme in artificial starch synthesis. Here, we report the crystal structure of a methylotrophic yeast DAK from Komagataella phaffii (formly Pichia pastoris, PpDAK). ATP was observed at a non-canonical site distinct from the canonical bacterial ATP-binding pocket. Docking further suggested that the canonical pocket remains accessible, indicating flexibility in ATP recognition. Sequence and phylogenetic analyses show that PpDAK clusters within a distinct methylotrophic yeast lineage and that residues surrounding the non-canonical ATP-binding site are conserved among methylotrophic yeasts. In addition, Mg2+ ions were identified in some substrate-binding pockets and docking suggested substantial overlap between Mg2+ and the predicted DHA-binding position. Together, these findings provide structural insights into ATP recognition in fungal DAKs and a framework for future functional studies and enzyme engineering.