Research paperFunctional characterization of cinnamate 4-hydroxylase from Helianthus annuus Linn using a fusion protein method
Introduction
Sunflower (Helianthus annuus L.) is an annual, bee-pollinated plant domesticated around 4000 BP in North America (Putt, 1997). As an essential oil crop and dried fruit, sunflower has an important economic and ornamental value. Sunflower is both a symbol of society and a major research focus of scientists. The researches on the chemical constituents of sunflowers started very early. A variety of compounds with complex structures isolated from sunflowers mainly included flavonoids, as well as some lignin, terpenoids and coumarin. As a wide class of plant polyphenols, flavonoids gradually draw the investigator's attention to the account of various biological activities (Sarbu et al., 2019). Flavonoids, which act as signal molecules, phytoalexins, allelochemicals, antibacterial defense compounds, and antidote, can protect plants from different biotic and abiotic stresses, such as UV damage and mechanical injury (Havsteen, 2002). Recent work that has provided some genomic information for sunflowers makes it easier to study the polyphenols biosynthesis process (Badouin et al., 2017). In short, based on the important role of secondary metabolites of the phenylpropanoid pathway, such as phenolic compounds in plant physiology, it is important to study the phenylpropanoid pathway related to the synthetic pathway of phenolic compounds in sunflower.
Cinnamate 4-hydroxylase (C4H) is the second key enzyme in the phenylpropanoid pathway. The key reactions in the phenylpropanoid pathway produce important secondary metabolites involved in plant development, disease resistance, and defense responses (Dixon and Paiva, 1995). The phenylpropanoid pathway involves three enzymes: firstly, L-phenylalanine is deaminated to produce trans-cinnamic acid (t-CA) by the action of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) (Lois et al., 1989). Subsequently, the formation of p-coumaric acid under the action of cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11) (Hubner et al., 2003). Finally, the conversion of p-coumaric acid to 4-Coumaroyl-CoA catalyzed by 4-coumarate: coenzyme A ligase (4CL, EC 6.2.1.12) (Vogt, 2010). 4-Coumaroyl-CoA is the precursor for many phenylpropanoid compounds including flavonoids, coumarins, and lignin (Jürgen Ehlting, 2006) (Fig. 1). C4H is a member of the CYP73 family that belongs to cytochrome P450 monooxygenases (CYP450s) (Chapple, 1998), which was discovered early in plants and was one of the typical P450s identified in plants (Nair and Vining, 1965, Russell and Conn, 1967). So, this enzyme is the most fully studied in plant cytochrome P450s and shows high specificity of its substrate trans-cinnamic acid (Kumar et al., 2013, Park et al., 2010, Tuan et al., 2010, Xia et al., 2017, Xu et al., 2010). The conversion of other structurally closely related compounds cannot occur or have low conversion efficiency. In general, trans-conformation and the presence of the carboxy group are essential for efficient catalysis (Werck-Reichhart, 1995).
Since the functional expression of CYP450s directly in the prokaryotic expression system was difficult. In the initial experiments, the proteins were mainly expressed in the modified yeast cells overexpressing CYP450 reductase (CPR, EC 1.6.2.4) (Pierrel et al., 1994, Urban et al., 1997, Urban et al., 1994). When the cytochrome P450 enzymes catalyze a lot of oxidation reactions it needs a CPR as a redox partner for supplying two electrons from NADPH through the FAD and FMN cofactors (Vermilion et al., 1981). CPR is confirmed to be present in most organisms, such as yeasts, plants, and animals. The function of CPR is to provide electrons to CYP450s to support P450-catalyzed oxidation reactions (Lee et al., 2014, Ro et al., 2002, Yang et al., 2010). In addition, the CYP450s and CPR are supposed to commonly localized at the cytoplasmic side of the endoplasmic reticulum (ER) membrane (Achnine et al., 2004). The N-terminus of both CYP450s and CPR contains a hydrophobic membrane-binding domain, which makes the expression of these proteins in prokaryotic microbes difficult because of low solubility. In short, the functional expression of CYP450s is difficult in prokaryotic microbes such as E. coli which lacks necessary organelles and CPR to support the function of plant CYP450s (Leonard et al., 2006). However, if the hydrophobic membrane-binding domain of CYP450s and CPR is removed and the fusion protein is expressed in E. coli to transfer electrons from NADPH, this problem can be solved (Leonard et al., 2006).
Although some studies have shown that some CYP450s, especially some C4H, can be correctly functionalized in prokaryotic microbes (Kim et al., 2009, Leonard et al., 2006, Li et al., 2018). However, it was difficult to express large fusion proteins in prokaryotic expression systems. Therefore, in some studies, only in vivo activity verification was performed (Li et al., 2018). So, it was still necessary to improve generic fusion protein methods for expressing various P450s in E. coli, due to the complexity of functional expression in yeast, and the difficulty of expressing large fusion proteins in prokaryotic expression systems and the difficulty of in vitro activity exploration in prokaryotic expression systems. Sunflower as an important plant, using the improved E. coli expression system to verify its C4H activity, and even using this method to explore the activity of other P450s related to important secondary metabolites of sunflower is very valuable.
In this study, we have screened and cloned three C4H genes in the current online access to the H. annuus (common sunflower) transcriptome and genomic database. We named it HaC4H1, HaC4H2, HaC4H3 and analyzed their expression profiles in different organs of sunflower and under various abiotic stresses. To validate the function of HaCHs, we improved an expression system containing modified Arabidopsis thaliana CYP450 redox partner ATR2 (belongs to CPR) by adding restriction sites and increasing linker. High-Performance Liquid Chromatography-Electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis showed these three enzymes catalyzed the formation of p-coumaric acid. Meanwhile, the in vitro activity was successfully verified on the basis of concentration and purification. Subsequently, to clarify whether this fusion protein method can be applicable to the expression of C4H from other plants in E. coli, we used this expression vector to verify the activity of PpC4H (C4H from Peucedanum praeruptorum) and AdC4H (C4H from Angelica decursiva) and they can also convert trans-cinnamic acid to p-coumaric acid.
Section snippets
Plant materials and chemicals
The seeds of The H. annuus are freshly harvested from the Xinjiang province of China. Sunflowers were grown in a light incubator with a long light cycle of 16 h light and 8 h dark every 24 h, the temperature was maintained at 30 ℃, the relative humidity was 50–65% and the light intensity was 3000 lx. Two months after plant germination, two-month-old seedlings were used for subsequent experiments.
Clone of HaC4Hs, PpC4H, and AdC4H
For the functional characterization of HaC4Hs, total RNA was extracted from H. annuus leaf powder
Identification and cloning of the cDNA encoding C4H in H. annuus
Using C4H sequences reported previously and the genomic sequence (accession no. HanXRQr1.0) and the transcriptome database of H. annuus (download from https://www.sunflowergenome.org/transcriptome/), a BLAST search was conducted. According to the E-value of alignments and after removing redundancies, three C4Hs were identified and cloned. We named them HaC4H1, HaC4H2 and HaC4H3. The ORF lengths of HaC4H1 and HaC4H3 were 1518 bp (encoding 506 residues) but HaC4H2 were 1521 bp (encoding 507
Discussion and conclusion
Sunflower has a good meaning as an important cash crop and an ornamental plant. Therefore, studying the key enzymes in metabolic pathways in sunflower is very important to understand and even modify plant metabolic pathways. C4H is a key enzyme in the core phenylpropanoid pathway and is involved in the synthesis and regulation of downstream flavonoids, lignins, and coumarins (Werck-Reichhart, 1995). But in the initial experiments, the proteins were mainly expressed in the modified yeast (Jürgen
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by the “Double First-Class” University project (CPU2018GY08, China), the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (B18056), and the Drug Innovation Major Project (2018ZX09711-001-007)
References (45)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)The biochemistry and medical significance of the flavonoids
Pharmacol. Therapeut.
(2002)- et al.
Functional expression of cinnamate 4-hydroxylase from Ammi majus L
Phytochemistry
(2003) - et al.
Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum
Plant Physiol. Biochem.
(2014) - et al.
Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli
Metab. Eng.
(2006) - et al.
Cloning, functional characterization and site-directed mutagenesis of 4-coumarate: coenzyme A ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn
Front. Plant Sci.
(2017) - et al.
Cinnamic acid hydroxylase in spinach
Phytochemistry
(1965) - et al.
The cinnamic acid 4-hydroxylase of pea seedlings
Arch. Biochem. Biophys.
(1967) - et al.
Functional characterization and correlation analysis of phenylalanine ammonia-lyase (PAL) in coumarin biosynthesis from Peucedanum praeruptorum Dunn
Phytochemistry
(2019) - et al.
Deletion of a conserved tetrapeptide, Ppgp, in P450 2c2 results in loss of enzymatic-activity without a change in its cellular location
Arch. Biochem. Biophys.
(1993)
Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5
J. Biol. Chem.
Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase
J. Biol. Chem.
Phenylpropanoid biosynthesis
Mol Plant
Molecular cloning and characterization of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis
Bioresour. Technol.
Characterization of two NADPH: cytochrome P450 reductases from cotton (Gossypium hirsutum)
Phytochemistry
Elucidation of the biosynthesis pathway and heterologous construction of a sustainable route for producing umbelliferone
J Biol Eng
Colocalization of L-phenylalanine ammonia-lyase and cinnarnate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis
Plant Cell
The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution
Nature
Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases
Annu. Rev. Plant Physiol. Plant Mol. Biol.
Characterization and expression patterns of a cinnamate-4-hydroxylase gene involved in lignin biosynthesis and in response to various stresses and hormonal treatments in Ginkgo biloba
Acta Physiol. Plant
Stress-induced phenylpropanoid metabolism
Plant Cell
Confidence limits on phylogenies: an approach using the bootstrap
Evolution
Cited by (8)
Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase
2024, Zhongguo Zhongyao ZazhiMYB Transcription Factor OsC1<sup>PLSr</sup> Involves the Regulation of Purple Leaf Sheath in Rice
2023, International Journal of Molecular SciencesResearch Progress of Function and Biosynthesis of Coumarins
2022, China BiotechnologyHrip1 mediates rice cell wall fortification and phytoalexins elicitation to confer immunity against Magnaporthe oryzae
2022, Frontiers in Plant SciencePhenylpropanoid Metabolism Pathway in Plants
2022, Chinese Journal of Biochemistry and Molecular Biology