The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in the synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein, we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose, and UDP-GalNAc. Structural comparison with the homolog from Escherichia coli (EcWaaG) revealed five key differences in the sugar-binding pocket. Solution-state NMR analysis showed that WT PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither WT PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

Lipopolysaccharide, NMR, Pseudomonas aeruginosa, glycosyltransferase, X-ray crystallography, WaaG

SMILES errors: aXKdop(2-4)[xLAla?(1-2)[bDGlcp(1-2)aLRhap(1-6)aDGlcp(1-4),aDGlcp(1-6)bDGlcp(1-3)]aDGalpN(1-3)[Cm(1-7),P-6)]aXLDmanHepp(1-3)[P-4),xXEtN(1-P-P-2)]aXLDmanHepp(1-5)]aXKdop:
SMILES error: incorrect linkage or the number of question bonds formed by some residue exceeds the number of available positions for bonding

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in the synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein, we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose, and UDP-GalNAc. Structural comparison with the homolog from Escherichia coli (EcWaaG) revealed five key differences in the sugar-binding pocket. Solution-state NMR analysis showed that WT PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither WT PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

Lipopolysaccharide, NMR, Pseudomonas aeruginosa, glycosyltransferase, X-ray crystallography, WaaG

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

NCCOP(=O)(O)OP(=O)(O)O[C@@H]1[C@@H]([C@@H](O)CO)O[C@H](O[C@@H]2[C@H](O[C@]3(C(=O)O)C[C@@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O3)C[C@](O)(C(=O)O)O[C@@H]2[C@H](O)CO)[C@@H](O)[C@H]1O[C@H]1O[C@H]([C@@H](O)CO[C@H]2O[C@H]([C@@H](O)CO)[C@@H](O)[C@H](O)[C@@H]2O)[C@@H](OP(=O)(O)O)[C@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)[C@H]3O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@H]2O)[C@@H]1O

2128.581 g/mol (C₆₉H₁₂₀NO₆₇P₃)

The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1→2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1→2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1→2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

cell wall, glycosylation, Galactomannan, Mannosyltransferase, Aspergillus

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

OC[C@@H](O)[C@@H]1O[C@@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@H](CO)[C@@H]3O[C@@H](O[C@H](CO)[C@@H]4O[C@@H](OC[C@@H](O)[C@@H]5O[C@@H](O[C@H](CO)[C@@H]6O[C@@H](O[C@H](CO)[C@@H]7O[C@@H](OC[C@H]8OC(O)[C@@H](O[C@H]9O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]9O)[C@@H](O)[C@@H]8O)[C@H](O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

1477.284 g/mol (C₅₄H₉₂O₄₆)

The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1→2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1→2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1→2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

cell wall, glycosylation, Galactomannan, Mannosyltransferase, Aspergillus

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](CO)O[C@@H](O)[C@H](NC(C)=O)[C@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO[C@H]3O[C@H](CO[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H]3O)[C@@H](O)[C@H](O[C@H]3O[C@H](CO[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)[C@@H]2O)[C@@H]1O

2532.236 g/mol (C₉₄H₁₅₈N₂O₇₆)