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. 2018 Aug 16;475(15):2547-2557.
doi: 10.1042/BCJ20180172.

Evidence for Substrate-Assisted Catalysis in N-acetylphosphoglucosamine Mutase

Free PMC article

Evidence for Substrate-Assisted Catalysis in N-acetylphosphoglucosamine Mutase

Olawale G Raimi et al. Biochem J. .
Free PMC article


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 α-d-phosphohexomutase metalloenzyme superfamily and catalyzes 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 represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.

Keywords: Aspergillus fumigatus; catalysis; phosphohexomutase; reaction mechanism.

Conflict of interest statement

The Authors declare that there are no competing interests associated with the manuscript.


Figure 1.
Figure 1.. Schematic illustrations of AfAGM1 assays.
(A) Phosphoglucomutase assay: a coupled assay with glucose-6-phosphate dehydrogenase (G6PDH) using G-1P as a substrate. (B) Phosphoglucosamine mutase assay: a coupled assay with A. fumigatus UDP-N-acetylglucosamine pyrophosphorylase (AfUAP1) and pyrophosphatase using GlcNAc-6P as a substrate. (C) A reverse phosphoglucomutase assay: a coupled assay with Trypanosoma brucei UDP-glucose pyrophosphorylase (TbUGP) and pyrophosphatase using G-6P as a substrate.
Figure 2.
Figure 2.. Overall structure of AfAGM1.
(A) Overall crystal structure of one of the two monomers found in the asymmetric unit of AfAGM1–GlcNAc-6P–Mg2+ ternary complex. Domains 1, 2, 3, and 4 are coloured in red, blue, green, and brown, respectively. The carbon sticks of AfAGM1_pSer69 and GlcNAc-6P are coloured in yellow and grey, respectively. (B) Surface representation of the monomeric forms of AfAGM1 in complex with GlcNAc-6P and Glc-1,6-bisP that show different conformational states during catalysis (colours of the different domains are the same as above).
Figure 3.
Figure 3.. Active sites of AfAGM1 and CaAGM1 in complex with different ligands.
The active sites of AfAGM1–GlcNAc-6P–Mg2+ and AfAGM1_S69A–Glc-1,6-bisP complexes are compared with CaAGM1–GlcNAc-6P–Zn2+-phosphate (PDB 2DKC) and CaAGM1–GlcNAc-1P–Zn2+-phosphate (PDB 2DKD) complexes [2]. Carbon atoms of residues within the active site pocket and sugars are shown as grey and green sticks, respectively. Mg2+ and Zn2+ ions, and water molecules are shown as pink, yellow, and blue spheres, respectively. Hydrogen bond interactions between the protein and the ligands are shown as black dashed lines, while Mg2+ and Zn2+ co-ordinations are shown as brown dash lines. The |Fo| − |Fc|, φcalc electron density maps around the ligands are shown contoured at 2.5 σ and also the final 2|Fo| − |Fc| electron density map for the phosphorylated active Ser69 is shown, contoured at 1.0 σ. A black arrow indicates the inline angle of attack of 158° of 1-hydroxyl of GlcNAc-6P on the phosphate located in AfAGM1_pSer69.
Figure 4.
Figure 4.. Catalytic mechanism.
(A) Model of wild-type AfAGM1 active site in complex with Glc-1,6-bisP and magnesium. Colours are the same as Figure 3 and a hydrogen atom bound to Ser69 is shown in yellow. (B) Scheme of the proposed reaction mechanism of phosphoacetylglucosamine mutase.

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