EC Number | Cloned (Comment) | Organism |
---|---|---|
1.2.1.26 | recombinant expression of N-terminally His6-tagged wild-type and mutant KGSADH enzymes in Escherichia coli strain DH10beta | Azospirillum brasilense |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
1.2.1.26 | 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 |
1.2.1.26 | 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 |
1.2.1.26 | 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 |
1.2.1.26 | 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 |
1.2.1.26 | 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 |
1.2.1.26 | 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 |
1.2.1.26 | R334Q/A337R | random mutagenesis, the mutant shows increased activity with 3-hydroxypropionaldehyde and altered cofactor kinetics compared to the wild-type enzyme | Azospirillum brasilense |
1.2.1.26 | 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 |
EC Number | Inhibitors | Comment | Organism | Structure |
---|---|---|---|---|
1.2.1.26 | NADH | - |
Azospirillum brasilense |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.2.1.26 | 0.025 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.033 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.037 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 0.044 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 0.17 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 0.21 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 0.27 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.27 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 0.28 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.29 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.43 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.54 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.55 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 0.66 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 0.7 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 0.78 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 0.81 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 1.4 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 1.6 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 2.2 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 2.3 | - |
NADP+ | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.2.1.26 | 2-oxoglutaric semialdehyde + NAD(P)+ + H2O | Azospirillum brasilense | - |
2-oxoglutarate + NAD(P)H + H+ | - |
? | |
1.2.1.26 | succinate semialdehyde + NAD+ + H2O | Azospirillum brasilense | activity of EC 1.2.1.24 | succinate + NADH + H+ | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.2.1.26 | Azospirillum brasilense | Q1JUP4 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
1.2.1.26 | recombinant N-terminally His6-tagged wild-type and mutant KGSADH enzymes from Escherichia coli strain DH10beta by nickel affinity chromatography and ultrafiltration | Azospirillum brasilense |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.2.1.26 | 2-oxoglutaric semialdehyde + NAD(P)+ + H2O | - |
Azospirillum brasilense | 2-oxoglutarate + NAD(P)H + H+ | - |
? | |
1.2.1.26 | 3-hydroxypropionaldehyde + NAD+ | - |
Azospirillum brasilense | 3-hydroxypropionate + NADH + H+ | - |
? | |
1.2.1.26 | 3-hydroxypropionaldehyde + NADP+ | very low activity | Azospirillum brasilense | 3-hydroxypropionate + NADPH + H+ | - |
? | |
1.2.1.26 | butyraldehyde + NAD+ | - |
Azospirillum brasilense | butanoate + NADH + H+ | - |
? | |
1.2.1.26 | hexaldehyde + NAD+ | - |
Azospirillum brasilense | hexanoate + NADH + H+ | - |
? | |
1.2.1.26 | propionaldehyde + NAD+ | - |
Azospirillum brasilense | propionate + NADH + H+ | - |
? | |
1.2.1.26 | succinate semialdehyde + NAD+ + H2O | activity of EC 1.2.1.24 | Azospirillum brasilense | succinate + NADH + H+ | - |
? | |
1.2.1.26 | valeraldehyde + NAD+ | - |
Azospirillum brasilense | valerate + NADH + H+ | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.2.1.26 | AbKGSADH | - |
Azospirillum brasilense |
1.2.1.26 | aldehyde dehydrogenase | - |
Azospirillum brasilense |
1.2.1.26 | ALDH | - |
Azospirillum brasilense |
1.2.1.26 | alpha-ketoglutarate-semialdehyde dehydrogenase | - |
Azospirillum brasilense |
1.2.1.26 | araE | - |
Azospirillum brasilense |
1.2.1.26 | KGSADH | - |
Azospirillum brasilense |
1.2.1.26 | More | cf. EC 1.2.1.24 | Azospirillum brasilense |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.2.1.26 | 25 | - |
assay at | Azospirillum brasilense |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.2.1.26 | 4 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 4.4 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 4.5 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 4.7 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 4.8 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 5 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 5.9 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 6 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 6.1 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 6.7 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 6.8 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 7.9 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 8.6 | - |
NADP+ | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 11 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 12 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 12 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 15 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 15 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 18 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 19 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 20 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A442P/P444T | Azospirillum brasilense |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.2.1.26 | 8 | - |
assay at | Azospirillum brasilense |
EC Number | Cofactor | Comment | Organism | Structure |
---|---|---|---|---|
1.2.1.26 | 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 | |
1.2.1.26 | NADP+ | low activity with 3-HPA | Azospirillum brasilense |
EC Number | General Information | Comment | Organism |
---|---|---|---|
1.2.1.26 | additional information | residues E253 and C287 are predicted as the catalytic residues | Azospirillum brasilense |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.2.1.26 | 0.74 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 3.74 | - |
NADP+ | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 5.43 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 6.71 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 6.818 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 6.82 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 8.571 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 9.375 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 12.364 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 14.103 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 15.581 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 17.241 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 35.294 | - |
3-hydroxypropionaldehyde | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 57.14 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Azospirillum brasilense | |
1.2.1.26 | 66.67 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant K273A/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 67.86 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 74.07 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 164.87 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant K273A/R334Q/A337R/A442P/T443E/P444A | Azospirillum brasilense | |
1.2.1.26 | 178.79 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant A110S/K273A/R334Q/A337R/A442P/P444T | Azospirillum brasilense | |
1.2.1.26 | 179.55 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R | Azospirillum brasilense | |
1.2.1.26 | 192 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant R334Q/A337R/A442P/P444T | Azospirillum brasilense |