Cloned (Comment) | Organism |
---|---|
wild-type and mutants introduced into the pT7GFS vector. Plasmid encodes for the expression of GFS bearing N-terminal hexahistidine tags, expressed in Escherichia coli BL21 (DE3) | Escherichia coli K-12 |
Protein Variants | Comment | Organism |
---|---|---|
C109S | active site mutant, produces GDP-6-deoxy-D-altrose as its major product (75%) besides GDP-L-fucose (25%) out of GDP-4-keto-6-deoxy-D-mannose | Escherichia coli |
C109S | produces GDP-6-deoxy-D-altrose as its major product, thus C-3'' epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. Results in the appearance of a kinetic isotope effect when [3''-2H]-GDP-6-deoxy-4-ketomannose is used as a substrate. The mutant catalyzes a rapid wash-in of solvent derived deuterium into the C-5'' position of GDP-fucose in the presence of NADP+ | Escherichia coli K-12 |
H179Q | is inactive, readily catalyzes the wash-out of deuterium from the C-3'' position of [3''-2H]-GDP-6-deoxy-4-keto-mannose | Escherichia coli K-12 |
H179Q | the mutant catalyzes isotope exchange into starting material indicates that its active site has not been dramatically perturbed and it is still able to bind substrate and catalyze deprotonation events | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P32055 | - |
- |
Escherichia coli K-12 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
by metal affinity chromatography | Escherichia coli K-12 |
Storage Stability | Organism |
---|---|
-80°C, 3 months | Escherichia coli K-12 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
GDP-4-keto-6-deoxy-D-mannose + NADPH + H+ | - |
Escherichia coli K-12 | GDP-L-fucose + NADP+ | - |
? | |
GDP-4-keto-6-deoxy-D-mannose + NADPH + H+ | - |
Escherichia coli | GDP-L-fucose + NADP+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
GDP-4-keto-6-deoxy-D-mannose epimerase/reductase | - |
Escherichia coli K-12 |
GDP-4-keto-6-deoxy-D-mannose epimerase/reductase | - |
Escherichia coli |
GDP-fucose synthase | - |
Escherichia coli K-12 |
GDP-fucose synthase | - |
Escherichia coli |
GFS | - |
Escherichia coli K-12 |
GFS | - |
Escherichia coli |
GMER | - |
Escherichia coli K-12 |
GMER | - |
Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1 | - |
GDP-6-deoxy-4-keto-mannose | mutant C109S, isotope [5''-2H]-GDP-6-deoxy-4-keto-mannose | Escherichia coli K-12 | |
1 | - |
GDP-6-deoxy-4-keto-mannose | wild-type, isotope [3''-2H]-GDP-6-deoxy-4-keto-mannose | Escherichia coli K-12 | |
1 | - |
GDP-6-deoxy-4-keto-mannose | wild-type, isotope [5''-2H]-GDP-6-deoxy-4-keto-mannose | Escherichia coli K-12 | |
1.5 | - |
GDP-6-deoxy-4-keto-mannose | mutant C109S, isotope [3''-2H]-GDP-6-deoxy-4-keto-mannose | Escherichia coli K-12 |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADPH | - |
Escherichia coli K-12 | |
NADPH | - |
Escherichia coli |