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Literature summary for 1.2.1.26 extracted from

  • Park, Y.S.; Choi, U.J.; Nam, N.H.; Choi, S.J.; Nasir, A.; Lee, S.G.; Kim, K.J.; Jung, G.Y.; Choi, S.; Shim, J.Y.; Park, S.; Yoo, T.H.
    Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD+, for enhancing the production of 3-hydroxypropionic acid (2017), Sci. Rep., 7, 17155 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.2.1.26

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of N-terminally His6-tagged wild-type and mutant KGSADH enzymes in Escherichia coli strain DH10beta Azospirillum brasilense

Protein Variants

Protein Variants Comment Organism
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+ Azospirillum brasilense
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 Azospirillum brasilense
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+ Azospirillum brasilense
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+ Azospirillum brasilense
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 Azospirillum brasilense
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 Azospirillum brasilense
R334Q/A337R random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme Azospirillum brasilense
R334Q/A337R/A442P/P444T random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme Azospirillum brasilense

Inhibitors

Inhibitors Comment Organism Structure
NADH
-
 Azospirillum brasilense

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.025
-
 NAD+ pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
0.033
-
 NAD+ pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
0.037
-
 NAD+ pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
0.044
-
 NAD+ pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
0.17
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
0.21
-
 NAD+ pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
0.27
-
 NAD+ pH 8.0, 25°C, recombinant mutant A442P/P444T Azospirillum brasilense
0.27
-
 NAD+ pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A Azospirillum brasilense
0.28
-
 NAD+ pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T Azospirillum brasilense
0.29
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
0.43
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
0.54
-
 NADP+ pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
0.55
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
0.66
-
 NADP+ pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
0.7
-
 NADP+ pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
0.78
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A Azospirillum brasilense
0.81
-
 NADP+ pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
1.4
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A442P/P444T Azospirillum brasilense
1.6
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
2.2
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T Azospirillum brasilense
2.3
-
 NADP+ pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-oxoglutaric semialdehyde + NAD(P)+ + H2O Azospirillum brasilense
-
 2-oxoglutarate + NAD(P)H + H+
-
 ?
succinate semialdehyde + NAD+ + H2O Azospirillum brasilense activity of EC 1.2.1.24 succinate + NADH + H+
-
 ?

Organism

Organism UniProt Comment Textmining
Azospirillum brasilense Q1JUP4
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged wild-type and mutant KGSADH enzymes from Escherichia coli strain DH10beta by nickel affinity chromatography and ultrafiltration Azospirillum brasilense

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-oxoglutaric semialdehyde + NAD(P)+ + H2O
-
 Azospirillum brasilense 2-oxoglutarate + NAD(P)H + H+
-
 ?
3-hydroxypropionaldehyde + NAD+
-
 Azospirillum brasilense 3-hydroxypropionate + NADH + H+
-
 ?
3-hydroxypropionaldehyde + NADP+ very low activity Azospirillum brasilense 3-hydroxypropionate + NADPH + H+
-
 ?
butyraldehyde + NAD+
-
 Azospirillum brasilense butanoate + NADH + H+
-
 ?
hexaldehyde + NAD+
-
 Azospirillum brasilense hexanoate + NADH + H+
-
 ?
propionaldehyde + NAD+
-
 Azospirillum brasilense propionate + NADH + H+
-
 ?
succinate semialdehyde + NAD+ + H2O activity of EC 1.2.1.24 Azospirillum brasilense succinate + NADH + H+
-
 ?
valeraldehyde + NAD+
-
 Azospirillum brasilense valerate + NADH + H+
-
 ?

Synonyms

Synonyms Comment Organism
AbKGSADH
-
 Azospirillum brasilense
aldehyde dehydrogenase
-
 Azospirillum brasilense
ALDH
-
 Azospirillum brasilense
alpha-ketoglutarate-semialdehyde dehydrogenase
-
 Azospirillum brasilense
araE
-
 Azospirillum brasilense
KGSADH
-
 Azospirillum brasilense
More cf. EC 1.2.1.24 Azospirillum brasilense

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Azospirillum brasilense

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4
-
 NADP+ pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
4.4
-
 NADP+ pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
4.5
-
 NADP+ pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
4.7
-
 NADP+ pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
4.8
-
 NAD+ pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
5
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
5.9
-
 NAD+ pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
6
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
6.1
-
 NAD+ pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
6.7
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
6.8
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
7.9
-
 NAD+ pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
8.6
-
 NADP+ pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
11
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A Azospirillum brasilense
12
-
 NAD+ pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
12
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A442P/P444T Azospirillum brasilense
15
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
15
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T Azospirillum brasilense
18
-
 NAD+ pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A Azospirillum brasilense
19
-
 NAD+ pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T Azospirillum brasilense
20
-
 NAD+ pH 8.0, 25°C, recombinant mutant A442P/P444T Azospirillum brasilense

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Azospirillum brasilense

Cofactor

Cofactor Comment Organism Structure
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 Azospirillum brasilense
NADP+ low activity with 3-HPA Azospirillum brasilense

General Information

General Information Comment Organism
additional information residues E253 and C287 are predicted as the catalytic residues Azospirillum brasilense

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.74
-
 NADP+ pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
3.74
-
 NADP+ pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
5.43
-
 NADP+ pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
6.71
-
 NADP+ pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
6.818
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T Azospirillum brasilense
6.82
-
 NADP+ pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
8.571
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A442P/P444T Azospirillum brasilense
9.375
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
12.364
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
14.103
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A Azospirillum brasilense
15.581
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
17.241
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense
35.294
-
 3-hydroxypropionaldehyde pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
57.14
-
 NAD+ pH 8.0, 25°C, recombinant wild-type enzyme Azospirillum brasilense
66.67
-
 NAD+ pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A Azospirillum brasilense
67.86
-
 NAD+ pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T Azospirillum brasilense
74.07
-
 NAD+ pH 8.0, 25°C, recombinant mutant A442P/P444T Azospirillum brasilense
164.87
-
 NAD+ pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A Azospirillum brasilense
178.79
-
 NAD+ pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T Azospirillum brasilense
179.55
-
 NAD+ pH 8.0, 25°C, recombinant mutant R334Q/A337R Azospirillum brasilense
192
-
 NAD+ pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T Azospirillum brasilense