Cloned (Comment) | Organism |
---|---|
gene lgdA, phylogenetic analysis and tree, recombinant expression of His-tagged enzyme in Escherichia coli strains BL21(DE3) or B834(DE3), the latter results in expression of a selenomethionine-labeled enzyme | Paracoccus laeviglucosivorans |
Crystallization (Comment) | Organism |
---|---|
purified His-tagged wild-type and selenomethionine-labeled enzyme in apoform and in a ternary complex with NAD+ and L-glucono-1,5-lactone, and in complex with myo-inositol and scyllo-inosose, hanging-drop vapor-diffusion method, mixing of 0.002 ml of protein solution with 0.02 ml of reservoir solution containing 0.1 M sodium acetate pH 4.8, and 12% PEG 3350, 20°C, crystals are soaked for one minute with 25 mM of NAD+ and 100 mM substrate in the reservoir solution, X-ray diffraction structure deteramintion and analysis at 1.75-2.3 A resolution, the structures complexed with myo-inositol/NAD+ and scyllo-inosose/NAD+ are solved at 2.3 A and 2.0 A resolution, respectively. Molecular replacement using the Se-Met-structure as the search model, modeling. The crystal is soaked with L-glucose and the resulting structure is determined | Paracoccus laeviglucosivorans |
Protein Variants | Comment | Organism |
---|---|---|
D191A | site-directed mutagenesis | Paracoccus laeviglucosivorans |
H195A | site-directed mutagenesis | Paracoccus laeviglucosivorans |
H318A | site-directed mutagenesis, the kcat value for scyllo-inositol decreases resulting in a very weak activity compared to wild-type enzyme | Paracoccus laeviglucosivorans |
K106A | site-directed mutagenesis | Paracoccus laeviglucosivorans |
R178A | site-directed mutagenesis, the mutation increases the Km value by 10fold compared to wild-type and decreases the kcat value for scyllo-inositol as a substrate, the mutant shows reduced activity | Paracoccus laeviglucosivorans |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Paracoccus laeviglucosivorans | |
3.7 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
40.3 | - |
L-epi-2-inosose | pH 6.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
40.6 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant mutant R178A | Paracoccus laeviglucosivorans | |
53.3 | - |
myo-inositol | pH 9.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
154 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant mutant H318A | Paracoccus laeviglucosivorans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-epi-2-inosose + NADH + H+ | Paracoccus laeviglucosivorans | - |
myo-inositol + NAD+ | - |
r | |
myo-inositol + NAD+ | Paracoccus laeviglucosivorans | - |
L-epi-2-inosose + NADH + H+ | - |
r | |
scyllo-inositol + NAD+ | Paracoccus laeviglucosivorans | - |
2,4,6/3,5-pentahydroxycyclohexanone + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Paracoccus laeviglucosivorans | K7ZP76 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration | Paracoccus laeviglucosivorans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-epi-2-inosose + NADH + H+ | - |
Paracoccus laeviglucosivorans | myo-inositol + NAD+ | - |
r | |
additional information | the enzyme performs also reversible L-glucose oxidation, structural basis and kinetics, overview. Substrate binding structures, detailed overview. Pl-scyllo-IDH has a different regioselectivity compared to myo-IDH toward myo-inositol | Paracoccus laeviglucosivorans | ? | - |
- |
|
myo-inositol + NAD+ | - |
Paracoccus laeviglucosivorans | L-epi-2-inosose + NADH + H+ | - |
r | |
scyllo-inositol + NAD+ | - |
Paracoccus laeviglucosivorans | 2,4,6/3,5-pentahydroxycyclohexanone + NADH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
homotetramer | crystal structure analysis | Paracoccus laeviglucosivorans |
More | one Pl-scyllo-IDH subunit contains two domains. The N-terminal domain adopted a Rossmann fold motif for the binding of NAD+, and the C-terminal domain bound substrate. The C-terminal domain contains a large beta-sheet composed of 8 parallel and anti-parallel beta-strands. A long loop extending from G303-G321 is found here to be a characteristic feature of Pl-scyllo-IDH. This loop is involved in subunit interactions, as well as substrate recognition in the adjacent subunit, as described below. Other interactions involved in oligomer formation are mainly mediated by the 8-stranded beta-sheet in the C-terminal domain | Paracoccus laeviglucosivorans |
Synonyms | Comment | Organism |
---|---|---|
IgdA | - |
Paracoccus laeviglucosivorans |
LgdA | - |
Paracoccus laeviglucosivorans |
Pl-scyllo-IDH | - |
Paracoccus laeviglucosivorans |
scyllo-inositol dehydrogenase | - |
Paracoccus laeviglucosivorans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Paracoccus laeviglucosivorans |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.43 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant mutant H318A | Paracoccus laeviglucosivorans | |
8.1 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant mutant R178A | Paracoccus laeviglucosivorans | |
9.53 | - |
myo-inositol | pH 9.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
11.75 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
75 | - |
L-epi-2-inosose | pH 6.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
NADH oxidation | Paracoccus laeviglucosivorans |
9 | - |
NAD+ reduction | Paracoccus laeviglucosivorans |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | cofactor binding mode and structure analysis, overview | Paracoccus laeviglucosivorans | |
NAD+ | - |
Paracoccus laeviglucosivorans | |
NADH | - |
Paracoccus laeviglucosivorans |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the glucose-fructose oxidase/inositol dehydrogenase/microbial rhizopine-catabolizing (GFO/IDH/MocA) family | Paracoccus laeviglucosivorans |
metabolism | scyllo-inositol dehydrogenase is involved in the first step in the pathway that oxidizes L-glucose to produce L-glucono-1,5-lactone with concomitant reduction of NAD+ dependent manner | Paracoccus laeviglucosivorans |
additional information | in addition to the conserved catalytic residues (Lys106, Asp191, and His195), another residue, His318, located in the loop region of the adjacent subunit, is involved in substrate recognition. The Arg178 residue located in the flexible loop at the entrance of the catalytic site is also involved in substrate recognition, and plays an important role in accepting both L-glucose and inositols as substrates. The corresponding distances in the myo-inositol and scyllo-inosose complexes are 2.8 A and 3.1 A, respectively, and the relative positions of NAD+ and the inositols are the same as those seen in the lactone complex | Paracoccus laeviglucosivorans |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0093 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant mutant H318A | Paracoccus laeviglucosivorans | |
0.18 | - |
myo-inositol | pH 9.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
0.2 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant mutant R178A | Paracoccus laeviglucosivorans | |
1.86 | - |
L-epi-2-inosose | pH 6.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans | |
3.18 | - |
scyllo-Inositol | pH 9.0, 25°C, recombinant wild-type enzyme | Paracoccus laeviglucosivorans |