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Literature summary extracted from

  • Chao, N.; Li, N.; Qi, Q.; Li, S.; Lv, T.; Jiang, X.N.; Gai, Y.
    Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR (2017), Planta, 245, 61-75 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.44 gene CCR1, DNA and amino acid sequence determination and analysis of diverse isozymes from Populus tomentosa, phylogenetic analysis, recombinant expression of His-tagged isozyme CCR1 in Escherichia coli strain M15, subcloning in Escherichia coli strain DH5alpha Populus tomentosa
1.2.1.44 gene CCR7, DNA and amino acid sequence determination and analysis of diverse isozymes from Populus tomentosa, phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant isozymes CCR7 in Escherichia coli strain M15, subcloning in Escherichia coli strain DH5alpha Populus tomentosa

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.44 A132S site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Populus tomentosa
1.2.1.44 A132T site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Populus tomentosa
1.2.1.44 A43V site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Populus tomentosa
1.2.1.44 F155H site-directed mutagenesis, the mutant is only active with feruloyl-CoA as substrate in contrast to the wild-type enzyme Populus tomentosa
1.2.1.44 F155Y site-directed mutagenesis, the mutant exhibits greater catalytic efficiency for sinapoyl-CoA compared to the wild-type PtoCCR7 Populus tomentosa
1.2.1.44 H208M site-directed mutagenesis, inactive mutant Populus tomentosa
1.2.1.44 H208V site-directed mutagenesis, inactive mutant Populus tomentosa
1.2.1.44 H208Y site-directed mutagenesis, inactive mutant Populus tomentosa
1.2.1.44 L192M site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme Populus tomentosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.44 additional information
-
additional information Michaelis-Menten kinetics Populus tomentosa
1.2.1.44 0.00986
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 0.01431
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged wild-type enzyme Populus tomentosa
1.2.1.44 0.01595
-
4-coumaroyl-CoA pH 6.0, 40°C, recombinant His-tagged wild-type enzyme Populus tomentosa
1.2.1.44 0.01643
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A132T Populus tomentosa
1.2.1.44 0.01731
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A132S Populus tomentosa
1.2.1.44 0.01924
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa
1.2.1.44 0.01973
-
4-coumaroyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa
1.2.1.44 0.02376
-
caffeoyl-CoA pH 6.0, 40°C, recombinant His-tagged wild-type enzyme Populus tomentosa
1.2.1.44 0.03123
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155H Populus tomentosa
1.2.1.44 0.03494
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A43V Populus tomentosa
1.2.1.44 0.04102
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged wild-type enzyme Populus tomentosa
1.2.1.44 0.04295
-
caffeoyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa
1.2.1.44 0.04887
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A43V Populus tomentosa
1.2.1.44 0.07474
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant L192M Populus tomentosa
1.2.1.44 0.0933
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.1.44 4-coumaroyl-CoA + NADPH + H+ Populus tomentosa
-
4-coumaraldehyde + CoA + NADP+
-
?
1.2.1.44 caffeoyl-CoA + NADPH + H+ Populus tomentosa
-
caffealdehyde + CoA + NADP+
-
?
1.2.1.44 feruloyl-CoA + NADPH + H+ Populus tomentosa preferred substrate coniferaldehyde + CoA + NADP+
-
?
1.2.1.44 feruloyl-CoA + NADPH + H+ Populus tomentosa highly specific for coniferaldehyde + CoA + NADP+
-
?
1.2.1.44 sinapoyl-CoA + NADPH + H+ Populus tomentosa
-
sinapaldehyde + CoA + NADP+
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.44 Populus tomentosa A0A0F7EVN0
-
-
1.2.1.44 Populus tomentosa T1WWB5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.44 recombinant His-tagged enzyme CCR1 from Escherichia coli strain M15 by nickel affinity chromatography Populus tomentosa
1.2.1.44 recombinant His-tagged wild-type and mutant enzymes CCR7 from Escherichia coli strain M15 by nickel affinity chromatography Populus tomentosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.44 4-coumaroyl-CoA + NADPH + H+
-
Populus tomentosa 4-coumaraldehyde + CoA + NADP+
-
?
1.2.1.44 caffeoyl-CoA + NADPH + H+
-
Populus tomentosa caffealdehyde + CoA + NADP+
-
?
1.2.1.44 feruloyl-CoA + NADPH + H+ preferred substrate Populus tomentosa coniferaldehyde + CoA + NADP+
-
?
1.2.1.44 feruloyl-CoA + NADPH + H+ highly specific for Populus tomentosa coniferaldehyde + CoA + NADP+
-
?
1.2.1.44 additional information isozyme PtoCCR1 exhibits specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. Substrate specificity, active site, molecular docking and modeling, overview Populus tomentosa ?
-
?
1.2.1.44 additional information substrate specificity, active site, molecular docking and modeling, overview. A132 in CCR7 combined with the catalytic triad might comprisethe catalytic center. In CCR7, L192, F155, and H208 are the substrate-binding sites Populus tomentosa ?
-
?
1.2.1.44 sinapoyl-CoA + NADPH + H+
-
Populus tomentosa sinapaldehyde + CoA + NADP+
-
?

Subunits

EC Number Subunits Comment Organism
1.2.1.44 ? x * 37080, CCR7, sequence calculation Populus tomentosa
1.2.1.44 ? x * 37540, CCR1, sequence calculation Populus tomentosa

Synonyms

EC Number Synonyms Comment Organism
1.2.1.44 CCR
-
Populus tomentosa
1.2.1.44 cinnamoyl-coenzyme A reductase
-
Populus tomentosa
1.2.1.44 PtoCCR1
-
Populus tomentosa
1.2.1.44 PtoCCR7
-
Populus tomentosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.1.44 40
-
-
Populus tomentosa

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.2.1.44 10 60 activity range, profile overview, the enzyme is active even below 10°C Populus tomentosa
1.2.1.44 20 60 activity range, profile overview Populus tomentosa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.1.44 0.0101
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A132T Populus tomentosa
1.2.1.44 0.0142
-
4-coumaroyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa
1.2.1.44 0.0183
-
4-coumaroyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 0.0192
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A132S Populus tomentosa
1.2.1.44 0.0327
-
caffeoyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa
1.2.1.44 0.0362
-
caffeoyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 0.064
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 0.098
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A43V Populus tomentosa
1.2.1.44 0.135
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa
1.2.1.44 0.1409
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155H Populus tomentosa
1.2.1.44 0.18
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 0.8375
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant A43V Populus tomentosa
1.2.1.44 1.033
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 1.905
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant L192M Populus tomentosa
1.2.1.44 2.1
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged mutant F155Y Populus tomentosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.44 6
-
-
Populus tomentosa

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.2.1.44 4 9 activity range, profile overview Populus tomentosa
1.2.1.44 5 8 activity range, profile overview Populus tomentosa

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.44 NADPH the conserved motifs G-X-X-G-X-X-A and D-X-X-D are reported to be involved in NAD(P) binding and adenine binding pocket stabilization. In addition, the NADP specificity motif R(X)5K is identified, which is a key structure that distinguishes CCR from other NAD(H)-dependent SDRs Populus tomentosa

pI Value

EC Number Organism Comment pI Value Maximum pI Value
1.2.1.44 Populus tomentosa sequence calculation
-
6.04
1.2.1.44 Populus tomentosa sequence calculation
-
8.22

General Information

EC Number General Information Comment Organism
1.2.1.44 evolution the conserved motifs G-X-X-G-X-X-A and D-X-X-D are reported to be involved in NAD(P) binding and adenine binding pocket stabilization. In addition, the NADP specificity motif R(X)5K is identified, which is a key structure that distinguishes CCR from other NAD(H)-dependent SDRs. Sequence comparisons and phylogenetic analysis of Populus tometosa CCRs, overview. Of the 11 PtoCCR and PtoCCR-like proteins, PtoCCR1 and 7 each catalyze at least one substrate. PtoCCR1 is active only with feruloyl-CoA as a substrate, while PtoCCR7 accepts all hydroxycinnamoyl-CoA esters Populus tomentosa
1.2.1.44 malfunction the positively charged extended structure of His208 was important in stabilizing CoA, while the mutants (M208, V208, and Y208) were unable to assume this function Populus tomentosa
1.2.1.44 additional information three-dimensional structure analysis of wild-type and mutant enzymes, molecular docking and modeling, overview Populus tomentosa
1.2.1.44 additional information three-dimensional structure analysis, molecular docking and modeling, overview Populus tomentosa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.2.1.44 1.15
-
caffeoyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 1.28
-
4-coumaroyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 1.56
-
sinapoyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 18.26
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa
1.2.1.44 72.19
-
feruloyl-CoA pH 6.0, 40°C, recombinant His-tagged enzyme Populus tomentosa