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Literature summary 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

EC Number Application Comment Organism
1.1.1.195 synthesis potential exploitation of rationally engineered forms of CAD2 for the targeted modification of monolignol composition in transgenic plants Medicago truncatula

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.195 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
1.1.1.195 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

EC Number Protein Variants Comment Organism
1.1.1.195 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
1.1.1.195 K169A site-directed mutagenesis, inactive mutant Medicago truncatula
1.1.1.195 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
1.1.1.195 S130A site-directed mutagenesis, inactive mutant Medicago truncatula
1.1.1.195 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
1.1.1.195 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
1.1.1.195 Y165A site-directed mutagenesis, inactive mutant Medicago truncatula
1.1.1.195 Y165F site-directed mutagenesis, inactive mutant Medicago truncatula

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.195 0.0066
-
 coniferyl aldehyde wild-type Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.008
-
 p-coumaryl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.0085
-
 coniferyl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.0094
-
 p-coumaryl aldehyde wild-type Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.0109
-
 sinapyl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.235
-
 sinapyl aldehyde Mt-CAD2 mutant Y136F/F226A, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.279
-
 sinapyl aldehyde Mt-CAD2 mutant Y136F, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.283
-
 sinapyl aldehyde Mt-CAD2 mutant F226A, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.73
-
 sinapyl aldehyde wild-type Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.195 cinnamaldehyde + NADPH + H+ Medicago truncatula
-
 cinnamyl alcohol + NADP+
-
 r
1.1.1.195 cinnamyl alcohol + NADP+ Medicago truncatula
-
 cinnamaldehyde + NADPH + H+
-
 r
1.1.1.195 coniferyl alcohol + NADP+ Medicago truncatula
-
 coniferyl aldehyde + NADPH + H+
-
 r
1.1.1.195 coniferyl aldehyde + NADPH + H+ Medicago truncatula
-
 coniferyl alcohol + NADP+
-
 r
1.1.1.195 p-coumaryl alcohol + NADP+ Medicago truncatula
-
 p-coumaryl aldehyde + NADPH + H+
-
 r
1.1.1.195 sinapyl alcohol + NADP+ Medicago truncatula
-
 sinapyl aldehyde + NADPH + H+
-
 r
1.1.1.195 sinapyl aldehyde + NADPH + H+ Medicago truncatula
-
 sinapyl alcohol + NADP+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.195 Medicago truncatula
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.195 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
1.1.1.195 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.195 cinnamaldehyde + NADPH + H+
-
 Medicago truncatula cinnamyl alcohol + NADP+
-
 r
1.1.1.195 cinnamyl alcohol + NADP+
-
 Medicago truncatula cinnamaldehyde + NADPH + H+
-
 r
1.1.1.195 coniferyl alcohol + NADP+
-
 Medicago truncatula coniferyl aldehyde + NADPH + H+
-
 r
1.1.1.195 coniferyl aldehyde + NADPH + H+
-
 Medicago truncatula coniferyl alcohol + NADP+
-
 r
1.1.1.195 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 ?
-
 ?
1.1.1.195 p-coumaryl alcohol + NADP+
-
 Medicago truncatula p-coumaryl aldehyde + NADPH + H+
-
 r
1.1.1.195 sinapyl alcohol + NADP+
-
 Medicago truncatula sinapyl aldehyde + NADPH + H+
-
 r
1.1.1.195 sinapyl aldehyde + NADPH + H+
-
 Medicago truncatula sinapyl alcohol + NADP+
-
 r

Subunits

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

Synonyms

EC Number Synonyms Comment Organism
1.1.1.195 CAD
-
 Medicago truncatula
1.1.1.195 Mt-CAD1
-
 Medicago truncatula
1.1.1.195 Mt-CAD2
-
 Medicago truncatula

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.195 30
-
 aldehyde reduction, assay at Medicago truncatula

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.195 0.024
-
 sinapyl aldehyde Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.039
-
 sinapyl aldehyde Mt-CAD2 mutant Y136F, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.044
-
 sinapyl aldehyde Mt-CAD2 mutant F226A, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.083
-
 sinapyl aldehyde Mt-CAD2 mutant Y136F/F226A, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.1
-
 coniferyl aldehyde Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.46
-
 p-coumaryl aldehyde Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 9.1
-
 coniferyl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 10.2
-
 sinapyl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 15.6
-
 p-coumaryl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.195 6.25
-
 aldehyde reduction, assay at Medicago truncatula

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.195 NADP+
-
 Medicago truncatula
1.1.1.195 NADPH
-
 Medicago truncatula

General Information

EC Number General Information Comment Organism
1.1.1.195 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
1.1.1.195 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
1.1.1.195 metabolism cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase are key enzymes of monolignol biosynthesis Medicago truncatula
1.1.1.195 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
1.1.1.195 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.195 0.0328
-
 sinapyl aldehyde Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.14
-
 sinapyl aldehyde Mt-CAD2 mutant Y136F, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.156
-
 sinapyl aldehyde Mt-CAD2 mutant F226A, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 0.354
-
 sinapyl aldehyde Mt-CAD2 mutant Y136F/F226A, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 15.3
-
 coniferyl aldehyde Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 48.9
-
 p-coumaryl aldehyde Mt-CAD2, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 926
-
 sinapyl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 1070
-
 coniferyl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula
1.1.1.195 1950
-
 p-coumaryl aldehyde Mt-CAD1, pH and temperature not specified in the publication Medicago truncatula