In vivo phosphorylation and in vitro autophosphorylation-inactivation of Kluyveromyces lactis hexokinase KlHxk1.

•The bifunctional hexokinase KlHxk1 is phosphorylated in vivo at serine-15.•KlHxk1 is partially inactivated in vitro by autophosphorylation at serine-157.•Crystallized phosphoserine-15 KlHxk1 exhibits a symmetrical dimeric structure.•Phosphorylation at serine-15 destabilizes the KlHxk1 homodimer in solution.•KlHxk1 phosphorylation at serine-15 presumably contributes to glucose signalling.