Evidence for substrate assisted catalysis in N-acetylphosphoglucosamine mutase

N -acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the a-D-phosphohexomutase metalloenzyme superfamily and catalyses the interconversion of N -acetylglucosamine-6-phosphate (GlcNAc-6P) to N -acetylglucosamine-1-phosphate (GlcNAc-1P) through N -acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown. Here we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg2+, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represent key snapshots along the reaction coordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.