Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) cells | Oryctolagus cuniculus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme in both apo form and NADH-bound holo form, X-ray diffraction structure determination and analysis at 1.70-1.85 A resolution | Oryctolagus cuniculus |
Protein Variants | Comment | Organism |
---|---|---|
D36R | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Oryctolagus cuniculus |
D36R/Q41N | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Oryctolagus cuniculus |
E97Q | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Oryctolagus cuniculus |
Q41N | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Oryctolagus cuniculus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
NAD+ | wild-type enzyme, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.018 | - |
L-gulonate | with NAD+, wild-type enzyme, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.028 | - |
NAD+ | mutant enzyme Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.054 | - |
NAD+ | mutant enzyme E97Q, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.12 | - |
NADP+ | mutant enzyme D36R/Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.2 | - |
NADP+ | mutant enzyme D36R, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.67 | - |
NADP+ | wild-type enzyme, pH 7.0, 25°C | Oryctolagus cuniculus | |
0.86 | - |
NADP+ | mutant enzyme Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
1.3 | - |
NAD+ | mutant enzyme D36R, pH 7.0, 25°C | Oryctolagus cuniculus | |
1.3 | - |
L-gulonate | with NAD+, mutant enzyme Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
1.6 | - |
L-gulonate | with NAD+, mutant enzyme E97Q, pH 7.0, 25°C | Oryctolagus cuniculus | |
2.4 | - |
L-gulonate | with NADP+, wild-type enzyme, pH 7.0, 25°C | Oryctolagus cuniculus | |
2.6 | - |
NAD+ | mutant enzyme D36R/Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
2.6 | - |
L-gulonate | with NADP+, mutant enzyme D36R/Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
3.3 | - |
L-gulonate | with NAD+, mutant enzyme D36R/Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
14 | - |
L-gulonate | with NAD+, mutant enzyme D36R, pH 7.0, 25°C | Oryctolagus cuniculus | |
14 | - |
L-gulonate | with NADP+, mutant enzyme Q41N, pH 7.0, 25°C | Oryctolagus cuniculus | |
18 | - |
L-gulonate | with NADP+, mutant enzyme D36R, pH 7.0, 25°C | Oryctolagus cuniculus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
36000 | - |
2 * 36000, recombinant enzyme, SDS-PAGE | Oryctolagus cuniculus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-gulonate + NAD+ | Oryctolagus cuniculus | - |
3-dehydro-L-gulonate + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Oryctolagus cuniculus | P14755 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) cells | Oryctolagus cuniculus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
L-gulonate + NAD+ = 3-dehydro-L-gulonate + NADH + H+ | Cys125, Glu97, and Ser124 are putative coenzyme/substrate-binding residues, catalytic mechanism with induced-fit mechanism upon coenzyme binding and involving a network-based substrate recognition, overview | Oryctolagus cuniculus |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
lens | - |
Oryctolagus cuniculus | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-gulonate + NAD+ | - |
Oryctolagus cuniculus | 3-dehydro-L-gulonate + NADH + H+ | - |
r | |
L-gulonate + NAD+ | NAD+ is preferred as cofactor | Oryctolagus cuniculus | 3-dehydro-L-gulonate + NADH + H+ | - |
r | |
L-gulonate + NADP+ | NAD+ is preferred as cofactor | Oryctolagus cuniculus | 3-dehydro-L-gulonate + NADPH + H+ | - |
r | |
additional information | active site structure and substrate recognition, importance of the dimeric state of the enzyme, overview | Oryctolagus cuniculus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 36000, recombinant enzyme, SDS-PAGE | Oryctolagus cuniculus |
More | the enzyme possesses two domains, the N-terminal domain with a Rossmann fold and the C-terminal domain with a helical fold. In the N-terminal domain of the NADH-bound structure exist 11 coenzyme-binding residues and two distinct side-chain conformers of coenzyme binding residue Ser124. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. Domain and subunit interface structures, structure comparison with the human enzyme, overview | Oryctolagus cuniculus |
Synonyms | Comment | Organism |
---|---|---|
More | the enzyme belongs to the GDH/lambdaCRY family | Oryctolagus cuniculus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Oryctolagus cuniculus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Oryctolagus cuniculus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | cofactor binding mode, overview | Oryctolagus cuniculus | |
NADH | cofactor binding mode, overview | Oryctolagus cuniculus | |
NADP+ | cofactor binding mode, overview | Oryctolagus cuniculus | |
NADPH | cofactor binding mode, overview | Oryctolagus cuniculus |
General Information | Comment | Organism |
---|---|---|
physiological function | L-gulonate 3-dehydrogenase is a bifunctional dimeric protein that functions not only as an NAD+-dependent enzyme in the uronate cycle but also as a taxon-specific lambda-crystallin in rabbit lens | Oryctolagus cuniculus |