Figure 6
Figure 6. Superimposition of a model human Fs synthase threaded onto human ABO glycosyltransferase. This GTB (a GTB variant, PDB entry 2RJ7) contains UDP-Gal and an antigen acceptor derivative in the binding site. Furthermore, this enzyme structure has adopted a closed conformation where an internal loop (residue 180-200) and the C-terminus (residue 345-354) have folded over the active site. The threading and minimization was done using Moe and resulted in a model highly similar to the GTB structure. (A) The GTA structure (orange; PDB entry 1LZI) has been superimposed onto the human Fs synthase model (green), although this GTA structure has a disordered internal loop and C-terminus. UDP-Gal (gray) from the GTB mutant (PDB entry 2RJ7) was also superimposed onto the figure to highlight the probable location of the donor substrate. The catalytically important His301 in GTA/GTB is shown as sticks and potentially forms a hydrogen bond to the O6 position on the Gal of the UDP-Gal (yellow dashed line). In the human Fs synthase model the Arg296, which corresponds to and overlaps His301 in ABO transferase, is shown as sticks and has adopted a rotamer side chain conformation away from the Gal. In almost any other conformation, the Arg296 side chain would be clashing with either nearby residues in the enzyme itself or with the donor. The H-antigen acceptor from the GTB structure is shown as black sticks and the manganese ion as a purple sphere. (B) Close-up of the active site from panel A. A GalNAc molecule from the HIC-Up database (yellow) is superimposed onto the Gal without introducing any clashes to the molecule. Assuming the UDP-GalNAc binds in a similar way to the Fs synthase as UDP-Gal does to GTB, the acetamido group can be easily accommodated. One reason for this is that at the corresponding position of Met-266 in GTB (known to be important for UDP-Gal specificity by sterically preventing binding of UDP-GalNAc) there is a glycine in Fs synthase which leaves ample space for the acetamido group. Color scheme as described. (C) The threaded structure of the modeled human Fs synthase. Close-up of the active site showing the position of the Arg296 (green) versus the Gln296 mutant (light-brown) as it was modeled in Moe with the residue replacement introduced. It is evident that residue Gln296 in the mutant is more probable to form a hydrogen bond with O6 of the Gal which could explain why the enzyme goes from inactive to active when the mutant is introduced.

Superimposition of a model human Fs synthase threaded onto human ABO glycosyltransferase. This GTB (a GTB variant, PDB entry 2RJ7) contains UDP-Gal and an antigen acceptor derivative in the binding site. Furthermore, this enzyme structure has adopted a closed conformation where an internal loop (residue 180-200) and the C-terminus (residue 345-354) have folded over the active site. The threading and minimization was done using Moe and resulted in a model highly similar to the GTB structure. (A) The GTA structure (orange; PDB entry 1LZI) has been superimposed onto the human Fs synthase model (green), although this GTA structure has a disordered internal loop and C-terminus. UDP-Gal (gray) from the GTB mutant (PDB entry 2RJ7) was also superimposed onto the figure to highlight the probable location of the donor substrate. The catalytically important His301 in GTA/GTB is shown as sticks and potentially forms a hydrogen bond to the O6 position on the Gal of the UDP-Gal (yellow dashed line). In the human Fs synthase model the Arg296, which corresponds to and overlaps His301 in ABO transferase, is shown as sticks and has adopted a rotamer side chain conformation away from the Gal. In almost any other conformation, the Arg296 side chain would be clashing with either nearby residues in the enzyme itself or with the donor. The H-antigen acceptor from the GTB structure is shown as black sticks and the manganese ion as a purple sphere. (B) Close-up of the active site from panel A. A GalNAc molecule from the HIC-Up database (yellow) is superimposed onto the Gal without introducing any clashes to the molecule. Assuming the UDP-GalNAc binds in a similar way to the Fs synthase as UDP-Gal does to GTB, the acetamido group can be easily accommodated. One reason for this is that at the corresponding position of Met-266 in GTB (known to be important for UDP-Gal specificity by sterically preventing binding of UDP-GalNAc) there is a glycine in Fs synthase which leaves ample space for the acetamido group. Color scheme as described. (C) The threaded structure of the modeled human Fs synthase. Close-up of the active site showing the position of the Arg296 (green) versus the Gln296 mutant (light-brown) as it was modeled in Moe with the residue replacement introduced. It is evident that residue Gln296 in the mutant is more probable to form a hydrogen bond with O6 of the Gal which could explain why the enzyme goes from inactive to active when the mutant is introduced.

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