Requirement of acidic amino acids in the glucuronyltransferase-P (GlcAT-P) flexible loop for its enzyme activity

GlcAT-P is a glucuronyltransferase that biosynthesizes the HNK-1 (Human Natural Killer-1) carbohydrate involved in synaptic plasticity. Previously, we purified the catalytic domain of recombinant human GlcAT-P and determined its X-ray crystal structure. In the GlcAT-P catalytic domain, a disordered region called a flexible loop, that showed no electron density, was identified; but not function for it has been established. Many glycosyltransferases have a similar flexible loop, and it was speculated that upon donor-substrate binding, the loop was ordered to partially facilitate acceptor binding, although it was unclear whether this notion was applicable to all glycosyltransferases. Here, we examined the role of the GlcAT-P flexible loop for its activity. A mutant GlcAT-P lacking the loop (GlcAT-PΔloop) hardly exhibited glucuronyltransferase activity in cells, and exhibited in vitro activity of less than 3% of wild-type, but Golgi-localization and dimerization were not influenced. Moreover, we revealed that GlcAT-PΔloop could bind with both acceptor- and donor-substrate same as wild-type. Finally, we demonstrated that the presence of acidic amino acids, but not amino acid length or sequence order within the GlcAT-P loop, was essential for enzyme activity. These results suggest that the flexible loop of GlcAT-P may have a specific function different from substrate binding.