Crystallization (Comment) | Organism |
---|---|
wild-type and mutant enzyme in complex with substrate UDP-alpha-D-galactopyranose or with inhibitor UDP, microbatch method at room temperature, for the substrate complex crystals: 10 mg/ml protein in 50 mM Tris, pH 8.0, 5 mM DTT, with 10 mM UDP-alpha-D-galactopyranose, and 10 mM dithionite, for the inhibitor complex crystals: 10 mg/ml protein in 25 mM Tris malonate, pH 8.0, and 10 mM UDP, for mutant enzyme-substrate complexes: 10 mg/ml protein in 25 mM Tris malonate, pH 8.0, with 15 mM UDP-alpha-D-galactopyranose, mixing of equal volumes of protein solution (with or without ligand) and crystallization solution and overlaid with oil, X-ray diffraction structure determination and analysis at 2.3-3.15 A resolution | Aspergillus fumigatus |
Protein Variants | Comment | Organism |
---|---|---|
R182A | site-directed mutagenesis, the mutant is almost inactive | Aspergillus fumigatus |
R182K | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Aspergillus fumigatus |
R327A | site-directed mutagenesis, inactive mutant | Aspergillus fumigatus |
R327K | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Aspergillus fumigatus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
UDP | - |
Aspergillus fumigatus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics of wild-type and mutant enzymes, overview | Aspergillus fumigatus | |
0.022 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R182K | Aspergillus fumigatus | |
0.0425 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, wild-type enzyme | Aspergillus fumigatus | |
0.0429 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R327K | Aspergillus fumigatus | |
0.607 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R182A | Aspergillus fumigatus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
UDP-alpha-D-galactopyranose | Aspergillus fumigatus | - |
UDP-alpha-D-galactofuranose | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aspergillus fumigatus | Q4W1X2 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
UDP-alpha-D-galactopyranose | - |
Aspergillus fumigatus | UDP-alpha-D-galactofuranose | - |
r |
Synonyms | Comment | Organism |
---|---|---|
UGM | - |
Aspergillus fumigatus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Aspergillus fumigatus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.079 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R182A | Aspergillus fumigatus | |
0.12 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R327K | Aspergillus fumigatus | |
0.44 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R182K | Aspergillus fumigatus | |
8.72 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, wild-type enzyme | Aspergillus fumigatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Aspergillus fumigatus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
flavin | the flavin needs to be reduced for the enzyme to be active | Aspergillus fumigatus |
General Information | Comment | Organism |
---|---|---|
evolution | conserved active site residues in Aspegillus fumigatus UGM compared to prokaryotic UGMs, overview | Aspergillus fumigatus |
additional information | Arg182 and Arg327 play important roles in stabilizing the position of the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis. Substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues Arg182 and Arg327, overview. The Aspergillus fumigatus enzyme contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor | Aspergillus fumigatus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00013 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R182A | Aspergillus fumigatus | |
0.003 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R327K | Aspergillus fumigatus | |
0.02 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, mutant R182K | Aspergillus fumigatus | |
0.205 | - |
UDP-alpha-D-galactopyranose | pH 7.0, 37°C, wild-type enzyme | Aspergillus fumigatus |