Application | Comment | Organism |
---|---|---|
synthesis | potential exploitation of rationally engineered forms of CAD2 for the targeted modification of monolignol composition in transgenic plants | Medicago truncatula |
Cloned (Comment) | Organism |
---|---|
diverse CAD or CAD-like genes in Medicago trunculata, phylogenetic analysis, recombinant expression of His-tagged Mt-CAD1 wild-type in Escherichia coli strain Rosetta2 | Medicago truncatula |
diverse CAD or CAD-like genes in Medicago trunculata, phylogenetic analysis, recombinant expression of His-tagged Mt-CAD2 wild-type and mutants in Escherichia coli strain Rosetta2 | Medicago truncatula |
Protein Variants | Comment | Organism |
---|---|---|
F226A | site-directed mutagenesis, the mutation leads to an enlarged phenolic binding site resulting in a 4fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2 | Medicago truncatula |
K169A | site-directed mutagenesis, inactive mutant | Medicago truncatula |
additional information | structure-based mutagenesis of Mt-CAD2 reveals and confirms the roles of key residues involved in catalysis and substrate binding and affords the engineering of catalytically active variants with increased turnover of sinapaldehyde | Medicago truncatula |
S130A | site-directed mutagenesis, inactive mutant | Medicago truncatula |
Y136F | site-directed mutagenesis, the mutation leads to an enlarged phenolic binding site resulting in a 10fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2 | Medicago truncatula |
Y136F/F226A | site-directed mutagenesis, the mutation leads to an enlarged phenolic binding site resulting in a 10fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2 | Medicago truncatula |
Y165A | site-directed mutagenesis, inactive mutant | Medicago truncatula |
Y165F | site-directed mutagenesis, inactive mutant | Medicago truncatula |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0066 | - |
coniferyl aldehyde | wild-type Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
0.008 | - |
p-coumaryl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
0.0085 | - |
coniferyl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
0.0094 | - |
p-coumaryl aldehyde | wild-type Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
0.0109 | - |
sinapyl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
0.235 | - |
sinapyl aldehyde | Mt-CAD2 mutant Y136F/F226A, pH and temperature not specified in the publication | Medicago truncatula | |
0.279 | - |
sinapyl aldehyde | Mt-CAD2 mutant Y136F, pH and temperature not specified in the publication | Medicago truncatula | |
0.283 | - |
sinapyl aldehyde | Mt-CAD2 mutant F226A, pH and temperature not specified in the publication | Medicago truncatula | |
0.73 | - |
sinapyl aldehyde | wild-type Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
cinnamaldehyde + NADPH + H+ | Medicago truncatula | - |
cinnamyl alcohol + NADP+ | - |
r | |
cinnamyl alcohol + NADP+ | Medicago truncatula | - |
cinnamaldehyde + NADPH + H+ | - |
r | |
coniferyl alcohol + NADP+ | Medicago truncatula | - |
coniferyl aldehyde + NADPH + H+ | - |
r | |
coniferyl aldehyde + NADPH + H+ | Medicago truncatula | - |
coniferyl alcohol + NADP+ | - |
r | |
p-coumaryl alcohol + NADP+ | Medicago truncatula | - |
p-coumaryl aldehyde + NADPH + H+ | - |
r | |
sinapyl alcohol + NADP+ | Medicago truncatula | - |
sinapyl aldehyde + NADPH + H+ | - |
r | |
sinapyl aldehyde + NADPH + H+ | Medicago truncatula | - |
sinapyl alcohol + NADP+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Medicago truncatula | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant Mt-CAD2 enzymes by nickel affinity chromatography, tag cleavage by thrombin, benzamidine affinity chromatography to remove thrombin, and gel filtration | Medicago truncatula |
recombinant His-tagged wild-type Mt-CAD1 enzyme by nickel affinity chromatography, tag cleavage by thrombin, benzamidine affinity chromatography to remove thrombin, and gel filtration | Medicago truncatula |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cinnamaldehyde + NADPH + H+ | - |
Medicago truncatula | cinnamyl alcohol + NADP+ | - |
r | |
cinnamyl alcohol + NADP+ | - |
Medicago truncatula | cinnamaldehyde + NADPH + H+ | - |
r | |
coniferyl alcohol + NADP+ | - |
Medicago truncatula | coniferyl aldehyde + NADPH + H+ | - |
r | |
coniferyl aldehyde + NADPH + H+ | - |
Medicago truncatula | coniferyl alcohol + NADP+ | - |
r | |
additional information | the enzyme Mt-CAD2 shows low activity with all substrates and compared to the activities of Mt-CAD1, substrate binding and specificity of Mt-CAD2, overview | Medicago truncatula | ? | - |
? | |
p-coumaryl alcohol + NADP+ | - |
Medicago truncatula | p-coumaryl aldehyde + NADPH + H+ | - |
r | |
sinapyl alcohol + NADP+ | - |
Medicago truncatula | sinapyl aldehyde + NADPH + H+ | - |
r | |
sinapyl aldehyde + NADPH + H+ | - |
Medicago truncatula | sinapyl alcohol + NADP+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
More | alpha-helical and beta-strand segments of Mt-CAD2, modelling, overview | Medicago truncatula |
Synonyms | Comment | Organism |
---|---|---|
CAD | - |
Medicago truncatula |
Mt-CAD1 | - |
Medicago truncatula |
Mt-CAD2 | - |
Medicago truncatula |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
aldehyde reduction, assay at | Medicago truncatula |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.024 | - |
sinapyl aldehyde | Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
0.039 | - |
sinapyl aldehyde | Mt-CAD2 mutant Y136F, pH and temperature not specified in the publication | Medicago truncatula | |
0.044 | - |
sinapyl aldehyde | Mt-CAD2 mutant F226A, pH and temperature not specified in the publication | Medicago truncatula | |
0.083 | - |
sinapyl aldehyde | Mt-CAD2 mutant Y136F/F226A, pH and temperature not specified in the publication | Medicago truncatula | |
0.1 | - |
coniferyl aldehyde | Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
0.46 | - |
p-coumaryl aldehyde | Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
9.1 | - |
coniferyl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
10.2 | - |
sinapyl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
15.6 | - |
p-coumaryl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.25 | - |
aldehyde reduction, assay at | Medicago truncatula |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Medicago truncatula | |
NADPH | - |
Medicago truncatula |
General Information | Comment | Organism |
---|---|---|
evolution | enzyme CAD2 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily, the SDR108E family together with a SDR115E daughter branch. Mt-CAD2 resides in the flowering plant phenylacetaldehyde-reductase subgroup. There are two CADs in Medicago truncatula, CAD1 and CAD2, which represent a classical and an atypical CAD belonging to the MDR and SDR families, respectively. Mt-CAD1 is highly active with all three substrates, coumaraldehyde, coniferaldehyde, and sinapaldehyde. By contrast, Mt-CAD2 exhibits relatively modest activity. The turnover rates (kcat) with coumaraldehyde, coniferaldehyde, and sinapaldehyde are only 3, 1, and 0.25%, respectively, of those for Mt-CAD1 | Medicago truncatula |
evolution | enzyme CAD2 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. There are two CADs in Medicago truncatula, CAD1 and CAD2, which represent a classical and an atypical CAD belonging to the MDR and SDR families, respectively. Mt-CAD1 is highly active with all three substrates, coumaraldehyde, coniferaldehyde, and sinapaldehyde. By contrast, Mt-CAD2 exhibits relatively modest activity. The turnover rates (kcat) with coumaraldehyde, coniferaldehyde, and sinapaldehyde are only 3, 1, and 0.25%, respectively, of those for Mt-CAD1 | Medicago truncatula |
metabolism | cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase are key enzymes of monolignol biosynthesis | Medicago truncatula |
additional information | the reaction mechanism involves a canonical SDR catalytic triad. Enzyme CAD2 shows substantial conformational flexibility, which plays an important role in the establishment of catalytically productive complexes of the enzyme with its NADPH and phenolic substrates. Mmolecular modeling and docking studies elucidate the specific interactions of Mt-CAD1 and Mt-CAD2 with NADPH and substrates, structural modeling of Mt-CAD1, overview | Medicago truncatula |
additional information | the reaction mechanism involves a canonical SDR catalytic triad. Enzyme CAD2 shows substantial conformational flexibility, which plays an important role in the establishment of catalytically productive complexes of the enzyme with its NADPH and phenolic substrates. Molecular modeling and docking studies elucidate the specific interactions of Mt-CAD1 and Mt-CAD2 with NADPH and substrates, binding pockets for NADP(H) co-substrate and phenolic-aldehyde substrate in Mt-CAD2, overview | Medicago truncatula |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0328 | - |
sinapyl aldehyde | Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
0.14 | - |
sinapyl aldehyde | Mt-CAD2 mutant Y136F, pH and temperature not specified in the publication | Medicago truncatula | |
0.156 | - |
sinapyl aldehyde | Mt-CAD2 mutant F226A, pH and temperature not specified in the publication | Medicago truncatula | |
0.354 | - |
sinapyl aldehyde | Mt-CAD2 mutant Y136F/F226A, pH and temperature not specified in the publication | Medicago truncatula | |
15.3 | - |
coniferyl aldehyde | Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
48.9 | - |
p-coumaryl aldehyde | Mt-CAD2, pH and temperature not specified in the publication | Medicago truncatula | |
926 | - |
sinapyl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
1070 | - |
coniferyl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula | |
1950 | - |
p-coumaryl aldehyde | Mt-CAD1, pH and temperature not specified in the publication | Medicago truncatula |