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Literature summary for 1.1.1.370 extracted from

  • Fukano, K.; Ozawa, K.; Kokubu, M.; Shimizu, T.; Ito, S.; Sasaki, Y.; Nakamura, A.; Yajima, S.
    Structural basis of L-glucose oxidation by scyllo-inositol dehydrogenase implications for a novel enzyme subfamily classification (2018), PLoS ONE, 13, e0198010 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Paracoccus laeviglucosivorans K7ZP76
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration Paracoccus laeviglucosivorans

Substrates and Products (Substrate)

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

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

Synonyms Comment Organism
IgdA
-
Paracoccus laeviglucosivorans
LgdA
-
Paracoccus laeviglucosivorans
Pl-scyllo-IDH
-
Paracoccus laeviglucosivorans
scyllo-inositol dehydrogenase
-
Paracoccus laeviglucosivorans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Paracoccus laeviglucosivorans

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
NADH oxidation Paracoccus laeviglucosivorans
9
-
NAD+ reduction Paracoccus laeviglucosivorans

Cofactor

Cofactor Comment Organism Structure
additional information cofactor binding mode and structure analysis, overview Paracoccus laeviglucosivorans
NAD+
-
Paracoccus laeviglucosivorans
NADH
-
Paracoccus laeviglucosivorans

General Information

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 [mM/s]

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