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Literature summary extracted from

  • Sattler, S.A.; Walker, A.M.; Vermerris, W.; Sattler, S.E.; Kang, C.
    Structural and biochemical characterization of cinnamoyl-CoA reductases (2017), Plant Physiol., 173, 1031-1044 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.44 gene CCR1, recombinant His-tagged enzyme expression in Escherichia coli strain Rosetta (DE3) Sorghum bicolor

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.1.44 purified recombinant His-tagged enzyme in complex with NADP+, hanging drop, vapor diffusion method, mixing of 20 mg/ml protein in 20 mM Tris base, pH 7.5, 2 mM DTT, and 1 mM NADP+, with an equal volume of reservoir solution , containing 100 mM Bis-Tris, pH 6.5, and 25% w/v PEG 3350, and equilibration against reservoir solution at 4°C, X-ray diffraction structure determination and analysis at 2.35 A resolution, modelling Sorghum bicolor

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.44 T154A the mutant enzyme displays significantly lower affinity for feruloyl-CoA compared with the wild-type enzyme Sorghum bicolor
1.2.1.44 T154Y the mutation in SbCCR1 leads to broader substrate specificity and faster turnover. The T154Y mutant exhibits 4.9 and 144fold increases in catalytic efficiency for feruloyl-CoA and 4-coumaroyl-CoA, respectively, over those of wild-type SbCCR1 Sorghum bicolor
1.2.1.44 Y310F the mutant enzyme displays significantly lower affinity for feruloyl-CoA compared with the wild-type enzyme Sorghum bicolor

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.44 additional information
-
 additional information thermodynamics and Michaelis-Menten kinetics Sorghum bicolor
1.2.1.44 0.014
-
 caffeoyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 0.063
-
 feruloyl-CoA pH 6.5, 30°C, mutant Y154Y Sorghum bicolor
1.2.1.44 0.07
-
 feruloyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 0.07
-
 4-coumaroyl-CoA pH 6.5, 30°C, mutant T154A Sorghum bicolor
1.2.1.44 0.1
-
 4-coumaroyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 0.113
-
 4-coumaroyl-CoA pH 6.5, 30°C, mutant Y154Y Sorghum bicolor
1.2.1.44 0.132
-
 feruloyl-CoA pH 6.5, 30°C, mutant Y310F Sorghum bicolor
1.2.1.44 0.155
-
 feruloyl-CoA pH 6.5, 30°C, mutant T154A Sorghum bicolor

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+ Sorghum bicolor
-
 4-coumaraldehyde + CoA + NADP+
-
 ?
1.2.1.44 caffeoyl-CoA + NADPH + H+ Sorghum bicolor
-
 caffealdehyde + CoA + NADP+
-
 ?
1.2.1.44 feruloyl-CoA + NADPH + H+ Sorghum bicolor best substrate coniferaldehyde + CoA + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.44 Sorghum bicolor C5YLL4
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.44 recombinant His-tagged enzyme CCR1 from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography, anion exchange chromatography, and dialysis Sorghum bicolor

Reaction

EC Number Reaction Comment Organism Reaction ID
1.2.1.44 cinnamaldehyde + CoA + NADP+ = cinnamoyl-CoA + NADPH + H+ although SbCCR1 displays higher affinity for caffeoyl-CoA or 4-coumaroyl-CoA than for feruloyl-CoA, the enzyme shows significantly higher activity for the latter substrate. In the first catalytic step, pro-R hydride transfer occurs from the C4 atom of NADPH to the reactive thioester carbonyl. The resulting oxyanion is temporarily stabilized by the oxyanion hole established from the side chain hydroxyl groups of Ser149 and Tyr183. Collapse of the tetrahedral intermediate is then followed by C-S bond cleavage and protonation of the CoA thiolate, in the presence of NADP+, there is very low affinity for the CoA ester compounds, which precludes the formation of a nonproductive complex, catalytic mechanism and substrate specificity, overview Sorghum bicolor
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.44 4-coumaroyl-CoA + NADPH + H+
-
 Sorghum bicolor 4-coumaraldehyde + CoA + NADP+
-
 ?
1.2.1.44 caffeoyl-CoA + NADPH + H+
-
 Sorghum bicolor caffealdehyde + CoA + NADP+
-
 ?
1.2.1.44 feruloyl-CoA + NADPH + H+ best substrate Sorghum bicolor coniferaldehyde + CoA + NADP+
-
 ?
1.2.1.44 additional information although SbCCR1 displays higher affinity for caffeoyl-CoA or 4-coumaroyl-CoA than for feruloyl-CoA, the enzyme shows significantly higher activity for the latter substrate. Substrate specificity, molecular docking, overview. Thr154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters Sorghum bicolor ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.2.1.44 More three-dimensional enzyme structure analysis and comparisons, overview Sorghum bicolor

Synonyms

EC Number Synonyms Comment Organism
1.2.1.44 CCR
-
 Sorghum bicolor
1.2.1.44 SbCCR1
-
 Sorghum bicolor

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.1.44 30
-
 assay at Sorghum bicolor

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.1.44 0.01
-
 caffeoyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 0.02
-
 4-coumaroyl-CoA pH 6.5, 30°C, mutant T154A Sorghum bicolor
1.2.1.44 0.05
-
 4-coumaroyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 0.47
-
 feruloyl-CoA pH 6.5, 30°C, mutant Y310F Sorghum bicolor
1.2.1.44 0.74
-
 feruloyl-CoA pH 6.5, 30°C, mutant T154A Sorghum bicolor
1.2.1.44 3.96
-
 feruloyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 8.42
-
 4-coumaroyl-CoA pH 6.5, 30°C, mutant Y154Y Sorghum bicolor
1.2.1.44 17.24
-
 feruloyl-CoA pH 6.5, 30°C, mutant Y154Y Sorghum bicolor

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.44 6.5
-
 assay at Sorghum bicolor

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.44 NADPH dependent on Sorghum bicolor

General Information

EC Number General Information Comment Organism
1.2.1.44 malfunction T154Y mutation in SbCCR1 leads to broader substrate specificity and faster turnover Sorghum bicolor
1.2.1.44 metabolism the enzyme is involved in the the monolignol biosynthetic pathway Sorghum bicolor
1.2.1.44 additional information the substrate-binding domain of the SbCCR1 is surrounded by two groups of a-helices, and the floor of the substrate-binding pocket is largely composed of beta-strands. Residues T154 and Y310 ae involved in substrate binding with ferulic acid, Tyr310 binds the 4-hydroxyl of feruloyl-CoA, while Thr154 binds the 3-methoxy group of this molecule. Molecular docking and modelling, overview Sorghum bicolor
1.2.1.44 physiological function cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-CoA esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. Isozyme SbCCR2 displays greater activity toward 4-coumaroyl-CoA than does isozyme SbCCR1, which implies a role in the synthesis of defense-related lignin. CCR1 is involved in lignification of stem tissues, whereas CCR2 is involved in lignification in response to attack by pathogens Sorghum bicolor

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 0.3
-
 4-coumaroyl-CoA pH 6.5, 30°C, mutant T154A Sorghum bicolor
1.2.1.44 0.5
-
 4-coumaroyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 0.8
-
 caffeoyl-CoA pH 6.5, 30°C, wild-type enzyme Sorghum bicolor
1.2.1.44 3.6
-
 feruloyl-CoA pH 6.5, 30°C, mutant Y310F Sorghum bicolor
1.2.1.44 4.8
-
 feruloyl-CoA pH 6.5, 30°C, mutant T154A Sorghum bicolor
1.2.1.44 56.6
-
 feruloyl-CoA pH 6.5, 30°C Sorghum bicolor
1.2.1.44 74.5
-
 4-coumaroyl-CoA pH 6.5, 30°C, mutant Y154Y Sorghum bicolor
1.2.1.44 273.6
-
 feruloyl-CoA pH 6.5, 30°C, mutant Y154Y Sorghum bicolor