Literature summary for 1.2.1.44 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 .

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
T154A	the mutant enzyme displays significantly lower affinity for feruloyl-CoA compared with the wild-type enzyme	Sorghum bicolor
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
Y310F	the mutant enzyme displays significantly lower affinity for feruloyl-CoA compared with the wild-type enzyme	Sorghum bicolor

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
4-coumaroyl-CoA + NADPH + H+	Sorghum bicolor	-	4-coumaraldehyde + CoA + NADP+	-	?
caffeoyl-CoA + NADPH + H+	Sorghum bicolor	-	caffealdehyde + CoA + NADP+	-	?
feruloyl-CoA + NADPH + H+	Sorghum bicolor	best substrate	coniferaldehyde + CoA + NADP+	-	?

Organism

Organism	UniProt	Comment	Textmining
Sorghum bicolor	C5YLL4	-	-

Purification (Commentary)

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

Reaction

Reaction	Comment	Organism	Reaction ID
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-coumaroyl-CoA + NADPH + H+	-	Sorghum bicolor	4-coumaraldehyde + CoA + NADP+	-	?
caffeoyl-CoA + NADPH + H+	-	Sorghum bicolor	caffealdehyde + CoA + NADP+	-	?
feruloyl-CoA + NADPH + H+	best substrate	Sorghum bicolor	coniferaldehyde + CoA + NADP+	-	?
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

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

Synonyms

Synonyms	Comment	Organism
CCR	-	Sorghum bicolor
SbCCR1	-	Sorghum bicolor

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
NADPH	dependent on	Sorghum bicolor

General Information

General Information	Comment	Organism
malfunction	T154Y mutation in SbCCR1 leads to broader substrate specificity and faster turnover	Sorghum bicolor
metabolism	the enzyme is involved in the the monolignol biosynthetic pathway	Sorghum bicolor
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
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]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.3	-	4-coumaroyl-CoA	pH 6.5, 30°C, mutant T154A	Sorghum bicolor
0.5	-	4-coumaroyl-CoA	pH 6.5, 30°C, wild-type enzyme	Sorghum bicolor
0.8	-	caffeoyl-CoA	pH 6.5, 30°C, wild-type enzyme	Sorghum bicolor
3.6	-	feruloyl-CoA	pH 6.5, 30°C, mutant Y310F	Sorghum bicolor
4.8	-	feruloyl-CoA	pH 6.5, 30°C, mutant T154A	Sorghum bicolor
56.6	-	feruloyl-CoA	pH 6.5, 30°C	Sorghum bicolor
74.5	-	4-coumaroyl-CoA	pH 6.5, 30°C, mutant Y154Y	Sorghum bicolor
273.6	-	feruloyl-CoA	pH 6.5, 30°C, mutant Y154Y	Sorghum bicolor