Figure 1.
Proposed biosynthetic pathway of UDP-galactofuranose (UDP-Galf) in Aspergillus nidulans.
Figure 2.
Electron density maps for UDP-Glcp modelled into the active site.
The σA-weighted 2|F o|-|F c| map (light purple) is contoured at 1σ while the σA-weighted |F o|-|F c| map (green) is contoured at 3σ. The C4 position of NAD+ has been denoted with a red arrow while the sugar C4 is marked with a blue arrow.
Figure 3.
Schematic of the initial HPLC studies for AnGALE.
a) Examination of UDP-Glcp/UDP-Galp substrate interconversion for wild-type and mutant enzymes. b) UNGM coupled reaction for monitoring interconversion of the UDP-GlcpNAc/UDP-GalpNAc substrate pair. Colors represent both product formation and the enzyme responsible for the catalytic reaction.
Figure 4.
Substrate interconversion within GALE occurs via a well-established three step process.
The C4 position of the sugar in each molecule is highlighted by a blue arrow.
Figure 5.
Stereoscopic ribbon depiction of a representative AnGALE monomeric unit.
Each subunit of the dimeric enzyme contains an N-terminal (red) and C-terminal (blue) domain. The co-factor and substrate appear in stick representations.
Figure 6.
Interactions anchoring the cofactor and substrate within the active site of AnGALE.
(a) Schematic and (b) model representations of the UDP-Glcp binding. (c) Schematic and (d) model representation of the NAD+ binding. For the schematic representations hydrogen bonds are shown in green and hydrophobic interactions in red. Hydrogen bonds are depicted as dashed lines in the model representations.
Figure 7.
Superposition of the monomeric ribbon representation for AnGALE (green) and HGALE (orange).
Cofactor and substrates for both enzymes are depicted in lighter variants as stick representations for clarity.
Figure 8.
Structure-based sequence alignment of A. nidulans, E. coli, and Human forms of GALE.
Identical residues are highlighted in red, similar residues are red surrounded with blue boxes.
Figure 9.
Competitive inhibition of GALE UDP-Galp activity by UDP-GalpNAc and UDP-GlcpNAc.
The apparent Ki for HGALE was taken from Reference [47].
Figure 10.
Validation of the docking procedure through reproduction of substrate binding modes.
C4 - C4 refers to the distance between C4(s) of nicotinamide and sugar, while Y-S refers to the distance between OH(s) of Y156 and sugar C4. The dashed red line represents the upper limit criteria (5 Å) for evaluating productive poses. PDB codes for docking UDP-Glcp: HGALE (1EK6) and EcGALE (1XEL); UDP-GlcpNAc: HGALE (1HZJ) and EcGALE (1LRJ). All units are in Å.