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2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
2,5-dioxopentanoate + NAD+ + H2O
2-oxoglutarate + NADH + H+
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
2-oxoglutaric semialdehyde + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H + H+
-
-
-
?
2-oxoglutaric semialdehyde + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
?
3-hydroxypropionaldehyde + NAD+
3-hydroxypropionate + NADH + H+
3-hydroxypropionaldehyde + NADP+
3-hydroxypropionate + NADPH + H+
very low activity
-
-
?
acetaldehyde + NAD(P)+ + H2O
acetate + NAD(P)H + H+
-
-
-
?
alpha-ketoglutaric semialdehyde + NAD(P)+ + H2O
alpha-ketoglutarate + NAD(P)H + H+
alpha-ketoglutaric semialdehyde + NADP+ + H2O
alpha-ketoglutarate + NADPH + H+
-
-
-
?
butyraldehyde + NAD(P)+ + H2O
n-butanoate + NAD(P)H + H+
-
-
-
-
?
butyraldehyde + NAD+
butanoate + NADH + H+
-
-
-
?
DL-glyceraldehyde + NADP+ + H2O
glycerate + NADPH + H+
glutaraldehyde + NAD(P)+ + H2O
glutarate + NAD(P)H + H+
-
-
-
?
glutaric semialdehyde + NADP+ + H2O
glutarate + NADPH
-
-
-
?
glycolaldehyde + NADP+ + H2O
glycolate + NADPH + H+
heptaldehyde + NAD(P)+ + H2O
n-heptanoate + NAD(P)H + H+
-
-
-
-
?
hexaldehyde + NAD+
hexanoate + NADH + H+
-
-
-
?
hexylaldehyde + NAD(P)+ + H2O
n-hexanoate + NAD(P)H + H+
-
-
-
-
?
octylaldehyde + NAD(P)+ + H2O
n-octanoate + NAD(P)H + H+
-
-
-
-
?
propanal + NAD(P)+ + H2O
propionate + NAD(P)H + H+
-
-
-
?
propionaldehyde + NAD(P)+ + H2O
propionate + NAD(P)H + H+
-
-
-
-
?
propionaldehyde + NAD+
propionate + NADH + H+
-
-
-
?
succinate semialdehyde + NAD(P)+ + H2O
? + NAD(P)H + H+
-
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
valeraldehyde + NAD(P)+ + H2O
n-pentanoate + NAD(P)H + H+
-
-
-
-
?
valeraldehyde + NAD+
valerate + NADH + H+
-
-
-
?
additional information
?
-
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
r
2,5-dioxopentanoate + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
r
2,5-dioxopentanoate + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
r
2,5-dioxopentanoate + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
-
r
2,5-dioxopentanoate + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
-
r
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
-
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
the enzyme is involved in the conversion of D-arabinose into the tricarboxylic acid cycle intermediate 2-oxoglutarate via the pentose oxidation pathway
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
the enzyme is specific for NADP+
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
the enzyme is specific for NADP+
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
-
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
the enzyme is involved in the conversion of D-arabinose into the tricarboxylic acid cycle intermediate 2-oxoglutarate via the pentose oxidation pathway
-
-
?
3-hydroxypropionaldehyde + NAD+
3-hydroxypropionate + NADH + H+
-
-
-
?
3-hydroxypropionaldehyde + NAD+
3-hydroxypropionate + NADH + H+
-
-
-
-
?
alpha-ketoglutaric semialdehyde + NAD(P)+ + H2O
alpha-ketoglutarate + NAD(P)H + H+
-
-
-
?
alpha-ketoglutaric semialdehyde + NAD(P)+ + H2O
alpha-ketoglutarate + NAD(P)H + H+
-
-
-
?
alpha-ketoglutaric semialdehyde + NAD(P)+ + H2O
alpha-ketoglutarate + NAD(P)H + H+
-
-
-
?
alpha-ketoglutaric semialdehyde + NAD(P)+ + H2O
alpha-ketoglutarate + NAD(P)H + H+
-
-
-
-
?
DL-glyceraldehyde + NADP+ + H2O
glycerate + NADPH + H+
-
-
-
?
DL-glyceraldehyde + NADP+ + H2O
glycerate + NADPH + H+
the enzyme is specific for NADP+
-
-
?
DL-glyceraldehyde + NADP+ + H2O
glycerate + NADPH + H+
the enzyme is specific for NADP+
-
-
?
DL-glyceraldehyde + NADP+ + H2O
glycerate + NADPH + H+
-
-
-
?
glycolaldehyde + NADP+ + H2O
glycolate + NADPH + H+
-
-
-
?
glycolaldehyde + NADP+ + H2O
glycolate + NADPH + H+
the enzyme is specific for NADP+
-
-
?
glycolaldehyde + NADP+ + H2O
glycolate + NADPH + H+
the enzyme is specific for NADP+
-
-
?
glycolaldehyde + NADP+ + H2O
glycolate + NADPH + H+
-
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
activity of EC 1.2.1.24
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
-
activity of EC 1.2.1.24
-
-
?
additional information
?
-
substrate and cofactor binding structures analysis, overview. The enzyme shows poor activity with NADP+ as a cofactor
-
-
-
additional information
?
-
-
substrate and cofactor binding structures analysis, overview. The enzyme shows poor activity with NADP+ as a cofactor
-
-
-
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2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
2-oxoglutaric semialdehyde + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H + H+
-
-
-
?
2-oxoglutaric semialdehyde + NAD+ + H2O
2-oxoglutarate + NADH + H+
-
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NAD(P)+ + H2O
2-oxoglutarate + NAD(P)H
-
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
the enzyme is involved in the conversion of D-arabinose into the tricarboxylic acid cycle intermediate 2-oxoglutarate via the pentose oxidation pathway
-
-
?
2,5-dioxopentanoate + NADP+ + H2O
2-oxoglutarate + NADPH + 2 H+
the enzyme is involved in the conversion of D-arabinose into the tricarboxylic acid cycle intermediate 2-oxoglutarate via the pentose oxidation pathway
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
activity of EC 1.2.1.24
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
-
activity of EC 1.2.1.24
-
-
?
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NAD+
-
-
NAD+
no significant preference between NAD+ and NADP+ in activity
NAD+
prefered cofactor of KGSADH-II
NAD+
NAD+-enzyme binding mode and structure, overview. The NAD+-binding pocket is constituted by seven loops (beta7-alpha4, beta8-alpha5, beta10-alpha7, beta11-beta12, alpha8-beta13, alpha9-alpha10, and alpha11-beta16) and four alpha-helices (alpha4, alpha6, alpha7, and alpha10). The adenine ring is stabilized in the hydrophobic pocket that is formed by Phe151, Pro211, Ala212, Phe229, Val235 and Leu239, and a hydrogen bond with Ser215 also contributes to the binding of the ring. Residues Lys178, Glu181, and Pro211 constitute a suitable space for binding of the ribose ring, and stabilize the 2'-hydroxyl-group of the ring. The formation of the ribose ring binding site does not seem to be large enough to accommodate the phosphorylated ribose ring. Therefore the enzyme shows poor activity with NADP+ as a cofactor. The diphosphate moiety is stabilized by residues Asn331, Arg333, and Arg334 through directly and water-mediated hydrogen bond networks. Residues Arg334 and Glu384 stabilize the ribose moiety of NAD+, and the nicotinamide ring is stabilized by residues Gln160 and Glu253 by hydrogen bonding
NAD+
preferred cofactor, residues chosen for generating the NAD+ binding pocket library are shown using the crystal structure of KGSADH complexed with NAD+ (PDB ID 5X5U), overview
NADP+
-
-
NADP+
alpha-KGSA dehydrogenase is an NADP+-preferring enzyme, since it is 58times more efficient with NADP+ than with NAD+
NADP+
prefered cofactor of KGSADH-III
NADP+
the enzyme is specific for NADP+
NADP+
low activity with 3-HPA
NADP+
the enzyme shows poor activity with NADP+ as a cofactor
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0.05 - 0.1
2,5-dioxopentanoate
-
-
0.17 - 2.2
3-hydroxypropionaldehyde
3.9
acetaldehyde
with NADP+ as cofactor
0.0065 - 0.5569
alpha-Ketoglutaric semialdehyde
0.1 - 0.31
Glutaraldehyde
0.016
Glutaric semialdehyde
-
-
0.37
glycolaldehyde
pH 6.0, 70°C, native enzyme form xylose-grown cells
0.45 - 1.66
succinate semialdehyde
0.17
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
0.29
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
0.43
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
0.55
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant R334Q/A337R
0.78
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A
1.4
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A442P/P444T
1.6
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant wild-type enzyme
2.2
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T
0.0065
alpha-Ketoglutaric semialdehyde
with NAD+ as cofactor
0.0075
alpha-Ketoglutaric semialdehyde
with NADP+ as cofactor
0.011
alpha-Ketoglutaric semialdehyde
with NADP+ as cofactor
0.014
alpha-Ketoglutaric semialdehyde
-
0.0246
alpha-Ketoglutaric semialdehyde
with NAD+ as cofactor
0.094
alpha-Ketoglutaric semialdehyde
-
with NADP+ as cofactor
0.5569
alpha-Ketoglutaric semialdehyde
-
with NAD+ as cofactor
0.1
Glutaraldehyde
with NADP+ as cofactor
0.31
Glutaraldehyde
with NAD+ as cofactor
0.025
NAD+
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
0.033
NAD+
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
0.037
NAD+
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
0.044
NAD+
pH 8.0, 25°C, recombinant mutant R334Q/A337R
0.21
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
0.27
NAD+
pH 8.0, 25°C, recombinant mutant A442P/P444T
0.27
NAD+
pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A
0.28
NAD+
pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T
0.0149
NADP+
-
0.54
NADP+
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
0.66
NADP+
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
0.7
NADP+
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
0.81
NADP+
pH 8.0, 25°C, recombinant mutant R334Q/A337R
2.3
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
0.3
propanal
with NADP+ as cofactor
1.05
propanal
with NAD+ as cofactor
0.45
succinate semialdehyde
with NAD+ as cofactor
1.66
succinate semialdehyde
with NADP+ as cofactor
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8.6
2,5-dioxopentanoate
pH 7.5, 70°C
5 - 15
3-hydroxypropionaldehyde
0.41
acetaldehyde
with NADP+ as cofactor
1.12 - 197
alpha-Ketoglutaric semialdehyde
4.8
DL-glyceraldehyde
pH 7.5, 70°C
0.65 - 4.13
Glutaraldehyde
5.3
glycolaldehyde
pH 7.5, 70°C
0.05 - 0.06
succinate semialdehyde
5
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
6
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
6.7
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
6.8
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant R334Q/A337R
11
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A
12
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A442P/P444T
15
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant wild-type enzyme
15
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T
1.12
alpha-Ketoglutaric semialdehyde
-
with NAD+ as cofactor
2.75
alpha-Ketoglutaric semialdehyde
-
with NADP+ as cofactor
15.4
alpha-Ketoglutaric semialdehyde
with NADP+ as cofactor
21.3
alpha-Ketoglutaric semialdehyde
with NAD+ as cofactor
23
alpha-Ketoglutaric semialdehyde
with NADP+ as cofactor
53.9
alpha-Ketoglutaric semialdehyde
with NAD+ as cofactor
197
alpha-Ketoglutaric semialdehyde
-
0.65
Glutaraldehyde
with NADP+ as cofactor
4.13
Glutaraldehyde
with NAD+ as cofactor
0.74
NAD+
-
-
4.8
NAD+
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
5.9
NAD+
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
6.1
NAD+
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
7.9
NAD+
pH 8.0, 25°C, recombinant mutant R334Q/A337R
12
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
18
NAD+
pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A
19
NAD+
pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T
20
NAD+
pH 8.0, 25°C, recombinant mutant A442P/P444T
1.08
NADP+
-
-
4
NADP+
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
4.4
NADP+
pH 8.0, 25°C, recombinant mutant R334Q/A337R
4.5
NADP+
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
4.7
NADP+
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
8.6
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
0.58
propanal
with NADP+ as cofactor
2.07
propanal
with NAD+ as cofactor
0.05
succinate semialdehyde
with NADP+ as cofactor
0.06
succinate semialdehyde
with NAD+ as cofactor
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
6.818 - 35.294
3-hydroxypropionaldehyde
6.818
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T
8.571
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A442P/P444T
9.375
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant wild-type enzyme
12.364
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant R334Q/A337R
14.103
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A
15.581
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
17.241
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
35.294
3-hydroxypropionaldehyde
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
57.14
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
66.67
NAD+
pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A
67.86
NAD+
pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T
74.07
NAD+
pH 8.0, 25°C, recombinant mutant A442P/P444T
164.87
NAD+
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
178.79
NAD+
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
179.55
NAD+
pH 8.0, 25°C, recombinant mutant R334Q/A337R
192
NAD+
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
0.74
NADP+
pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T
3.74
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
5.43
NADP+
pH 8.0, 25°C, recombinant mutant R334Q/A337R
6.71
NADP+
pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T
6.82
NADP+
pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A
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malfunction
low ALDH activity has been reported to cause intracellular accumulation of highly toxic 3-hydroxypropionaldehyde (3-HPA) seriously hampering the cell growth
metabolism
3-hydroxypropionate biosynthesis pathway from glycerol involves enzymes coenzyme B12-dependent glycerol dehydratase (DhaB) and aldehyde dehydrogenase (ALDH), overview. alpa-Ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH) can catalyze the ALDH reaction forming 3-hydroxypropionate
physiological function
alpa-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH) can catalyze the ALDH reaction forming 3-hydroxypropionate, but it is not its physiological substrate
additional information
molecular docking simulations of AbKGSADH with 2-oxoglutaric semialdehyde (alpha-KGSA) and succinate semialdehyde (SSA). Molecular docking simulations reveal that these two substrates fit well into the somewhat positively charged substrate binding pocket. The aldehyde-groups of these substrates, which are the sites of enzyme reaction, are located in the same place around the catalytic residues. The aldehyde-group of alpha-KGSA is stabilized by Gln160 and Arg163 through hydrogen bonds, and two catalytic residues, Glu253 and Cys287, also assist the binding of the molecule. The 4'-oxo-group of alpha-KGSA is stabilized by hydrogen bonds with Arg281, and the carboxyl-group of the molecule is stabilized by Glu106 and Gln160. The substrate binding pocket is also formed by several hydrophobic residues, such as Phe156, Val286, Ile288, Pro444, and Phe450, which seem to contribute to the stabilization of the hydrophobic part of alpha-KGSA. The binding of SSA is similar to that of alpha-KGSA, however, the stabilization of the carboxyl-group of SSA is quite different. Arg281, a residue that is involved in the stabilization of the 4'-oxo-group of alpha-KGSA, forms a hydrogen bond with the carboxyl-group of SSA instead. These observations explain how AbKGSADH can accommodate both alpha-KGSA and SSA as real substrates
additional information
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molecular docking simulations of AbKGSADH with 2-oxoglutaric semialdehyde (alpha-KGSA) and succinate semialdehyde (SSA). Molecular docking simulations reveal that these two substrates fit well into the somewhat positively charged substrate binding pocket. The aldehyde-groups of these substrates, which are the sites of enzyme reaction, are located in the same place around the catalytic residues. The aldehyde-group of alpha-KGSA is stabilized by Gln160 and Arg163 through hydrogen bonds, and two catalytic residues, Glu253 and Cys287, also assist the binding of the molecule. The 4'-oxo-group of alpha-KGSA is stabilized by hydrogen bonds with Arg281, and the carboxyl-group of the molecule is stabilized by Glu106 and Gln160. The substrate binding pocket is also formed by several hydrophobic residues, such as Phe156, Val286, Ile288, Pro444, and Phe450, which seem to contribute to the stabilization of the hydrophobic part of alpha-KGSA. The binding of SSA is similar to that of alpha-KGSA, however, the stabilization of the carboxyl-group of SSA is quite different. Arg281, a residue that is involved in the stabilization of the 4'-oxo-group of alpha-KGSA, forms a hydrogen bond with the carboxyl-group of SSA instead. These observations explain how AbKGSADH can accommodate both alpha-KGSA and SSA as real substrates
additional information
residues E253 and C287 are predicted as the catalytic residues
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A110S/K273A/A442P/P444T
random mutagenesis, the mutant shows reduced activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme, no activity with NADP+
A110S/K273A/R334Q/A337R/A442P/P444T
random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme
A442P/P444T
random mutagenesis, the mutant shows reduced activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme, no activity with NADP+
C287A
site-directed mutagenesis, the mutant is almost inactive
E253A
site-directed mutagenesis, the mutant is almost inactive
F156A
site-directed mutagenesis, the mutant is almost inactive
F450A
site-directed mutagenesis, the mutant is almost inactive
I288A
site-directed mutagenesis, the mutant is almost inactive
K273A/A442P/T443E/P444A
random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme, no activity with NADP+
K273A/R334Q/A337R/A442P/T443E/P444A
random mutagenesis, the mutant shows strongly increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme
N159A
site-directed mutagenesis, the mutant shows 50% increased activity compared to wild-type enzyme
Q160A
site-directed mutagenesis, the mutant shows 30% increased activity compared to wild-type enzyme
R163A
site-directed mutagenesis, the mutant shows 10% reduced activity compared to wild-type enzyme
R334Q/A337R
random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme
R334Q/A337R/A442P/P444T
random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme
V286A
site-directed mutagenesis, the mutant shows 50% reduced activity compared to wild-type enzyme
additional information
alpha-KGSA dehydrogenase activity in cell extracts from the mutant DELTAACIAD0131 grown on succinate as the sole carbon source is undetectable
additional information
engineering of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for prodduction of 3-hydroxypropanoate (HP) from 3-hydroxypropionaldehyde (3-HPA). A directed evolutionary strategy is adopted as the engineering approach for modifying the substrate-binding sites of KGSADH. The residues in the binding sites for the substrates, 3-HPA and NAD+, are randomized, and the resulting libraries are screened for higher activity. Isolated KGSADH variants have significantly lower Km values for both the substrates. The enzymes also show higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain expressing one of the engineered KGSADH variants exhibits less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that expressing the wild-type KGSADH. The flask culture of the Pseudomonas denitrificans strain with the mutant KGSADH results in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM). Mutant structure modeling, overview
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