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Literature summary for 1.1.1.195 extracted from

  • Pan, H.; Zhou, R.; Louie, G.V.; Muehlemann, J.K.; Bomati, E.K.; Bowman, M.E.; Dudareva, N.; Dixon, R.A.; Noel, J.P.; Wang, X.
    Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis (2014), Plant Cell, 26, 3709-3727.
    View publication on PubMedView publication on EuropePMC

Application

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(Commentary)

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Medicago truncatula
-
-
-

Purification (Commentary)

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 and Products (Substrate)

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

Subunits Comment Organism
More alpha-helical and beta-strand segments of Mt-CAD2, modelling, overview Medicago truncatula

Synonyms

Synonyms Comment Organism
CAD
-
Medicago truncatula
Mt-CAD1
-
Medicago truncatula
Mt-CAD2
-
Medicago truncatula

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
aldehyde reduction, assay at Medicago truncatula

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.25
-
aldehyde reduction, assay at Medicago truncatula

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Medicago truncatula
NADPH
-
Medicago truncatula

General Information

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 [mM/s]

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