Covalent bond formation between amino acids and lignin: Cross-coupling between proteins and lignin

doi:10.1016/j.phytochem.2013.09.012

Phytochemistry

Volume 96, December 2013, Pages 449-456

https://doi.org/10.1016/j.phytochem.2013.09.012 Get rights and content

Highlights

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The reaction of aromatic amino acids with intermediates of lignin biosynthesis was examined.
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Products formed from reaction of amino acids with intermediates of lignin biosynthesis were identified by mass spectroscopy.
•
Amino acid adducts were formed with Tyr, Thr and Cys through a reaction of the side-chain with the quinone methide.
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The cross-coupling was also shown to occur at the protein level.
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These results show how peptide linkages can be introduced into the lignin polymer.

Abstract

The present study characterized the products formed from the reaction of amino acids and in turn, proteins, with lignin resulting in cross-coupling. When added to reaction mixtures containing coniferyl alcohol, horseradish peroxidase and H₂O₂, three amino acids (Cys, Tyr, and Thr) are able to form adducts. The low molecular weight products were analyzed by HPLC and from each reaction mixture, one product was isolated and analyzed by LC/MS. LC/MS results are consistent with bond formation between the polar side-chain of these amino acids with Cα. These results are consistent with the cross-coupling of Cys, Tyr and Thr through a quinone methide intermediate. In addition to the free amino acids, it was found that the cross-coupling of proteins with protolignin through Cys or Tyr residues. The findings provide a mechanism by which proteins and lignin can cross-couple in the plant cell wall.

Graphical abstract

Lignin can cross-couple with the side-chain of amino acids, possibly through a quinone intermediate thereby creating covalent bonds between lignin and proteins.

Introduction

Lignocellulosic material is composed of three major constituents, cellulose, hemicellulose and lignin (Wyman, 1994). Cellulose is the most abundant of the components, accounting for approximately 50% of woody biomass (Bull, 1991). In contrast to the α-1,4 linkage of starch, the β-1,4 linkage causes cellulose to be highly crystalline (Taylor, 2008). Hemicellulose is more heterogeneous in structure than cellulose (Scheller and Ulvskov, 2010). Structurally, lignin is the most heterogeneous component of lignocellulose. It is the most abundant aromatic polymer in nature, accounting for approximately 30% of the carbon on earth (Boerjan et al., 2003).

The architecture of the plant cell wall is an active area of investigation due to the importance of lignocellulosic material as a source for bioethanol (Bull, 1991). These investigations have focused on determining the nature of the covalent bonds and non-covalent interactions which are intermolecular and intra-molecular. An example of an intramolecular interaction is the extensive hydrogen bonding within the cellulose polymer (Taylor, 2008). This renders cellulose highly crystalline and difficult to hydrolyze enzymatically (Chundawat et al., 2011). Less well defined are the inter-molecular bonds and interaction. To date, no evidence exists for covalent bonds between cellulose and hemicellulose. The covalent bond and non-covalent interactions between lignin and polysaccharides have been studied with “lignin-carbohydrate complex” (LCC) isolated from the plant cell walls (Jin et al., 2006, Morrison, 1974a, Morrison, 1974b, Neilson and Richards, 1982). The presence of covalent bonds between lignin and cellulose has been detected by NMR spectroscopic analysis of carboxymethylated LCC (Jin et al., 2006). In addition, studies in Lolium perenne (Morrison, 1974a, Morrison, 1974b) and bovine rumen (Neilson and Richards, 1982) have provided evidence of covalent coupling between hemicellulose and lignin. The proposed coupling to lignin is via the free hydroxyl groups of hemicelluloses which act as nucleophiles, attacking the quinone methide intermediate of lignin biosynthesis (Freudenberg, 1959, Helm, 2000, Leary, 1980). The quinone methide is an intermediate formed from every coupling that involves reaction through a β-position (Freudenberg, 1959). The electro-positive C-α of the quinone methide is susceptible to nucelophilic attack. When the nucleophile is water, the α-hydroxyl group is the resultant product.

Another component of the plant cell wall is protein. Proteins account for approximately 0.5% of the dry weight of the plant cell wall (Laidlaw and Smith, 1965, Martius, 1992). Research has shown that a large number of different proteins are cell wall-associated (Cassab, 1998). Some plant cell wall proteins may arise simply from the necessity of having proteins participate in plant cell wall synthesis. Other proteins may arise from the role of proteins in cell–cell interaction (Cheung et al., 1995, Rubinstein et al., 1995), morphogenesis (Bonilla et al., 1997), and plant differentiation (Knox et al., 1991, Kreuger and Vanholst, 1993). The ultimate fate of proteins in the cell wall is not well known. However, studies have suggested that wall-localized proteins become cross coupled with the wall as structural components (Cassab, 1998). That component may be lignin. Researchers have shown that protein contaminations are always found along with lignin isolations from plant tissue (Jamet et al., 2006). Another study by Keller et al. (1989), using immune-gold labeling localized the cell-wall associated glycine rich protein with lignified secondary cell wall. This is consistent with the glycine rich protein cross-coupling with lignin during cell wall formation (Keller et al., 1989).

The mechanism of this cross-coupling may be similar to that between hemicellulose and lignin, involving the quinone methide. In an in vitro study of peptide and synthetic lignin, researchers demonstrated that polylysine becomes embedded into the growing lignin polymer (McDougall et al., 1996). These researchers also showed that polylysine which also contained Tyr, enhances the association of peptides into lignin (McDougall et al., 1996). The chemical nature of the interactions between protein and other cell wall components are still undefined.

In this study, the reaction of amino acids to with the growing lignin polymer was examined. It was found that three amino acids (Cys, Tyr, and Thr) were covalently coupled with lignin when added to the coniferyl alcohol(1)/peroxidase reaction mixture. In addition, the capacity of proteins to cross-couple with lignin in vitro through Cys or through Tyr residues was demonstrated. These findings provide a mechanism by which proteins and lignin can be coupled in the plant cell wall.

Section snippets

Amino acid/coniferyl alcohol(1) cross-coupling

Transgenic plants were previously generated where proteins containing a high Tyr content were secreted into the lignifying plant cell wall (Liang et al., 2008). A protein with a high Tyr content was chosen because Tyr contains a phenolic group, similar to the lignin precursors. As such, it could potentially be coupled into lignin by a free radical coupling process. In the present study, the objective was to determine the mechanism of Tyr cross-coupling with lignin. Tyr was added to reaction

Discussion

Proteins have been shown to account for approximately 0.5% of the dry weight of the plant cell wall (Laidlaw and Smith, 1965, Martius, 1992). Although these proteins may have been proteins used for cell wall synthesis, very little is known about the origin of these proteins. The nature of the interactions between cell wall components and these proteins is also not well understood. The work of Whitmore (Whitmore, 1976, Whitmore, 1982) suggests that the cell wall-embedded proteins are coupled to

Conclusions

This work characterizes the cross-coupling between amino acids/proteins with lignin. The original strategy for cross-coupling proteins with lignin involved the use of Tyr in the hope that the phenolic substituent would cross-couple via a free radical mechanism. However, the present work shows that alternative mechanisms for cross-coupling may be operative. The data obtained is consistent with cross-coupling occurring through the quinone methide intermediate. This is suggested by the results

Chemicals

[¹⁴C]cystine (>9.17 × 10⁹ Bq/mmol), [³H]tyrosine (1.47–2.20 × 10¹² Bq/mmol), [¹⁴C]glycine (>3.67 × 10⁹ Bq/mmol), and [³H]sodium borohydride (1.84–5.51 × 10¹¹ Bq/mmol) were purchased from PerkinElmer (MA, USA). Horseradish peroxidase (HRP) Type II and tris (2-carboxyethyl) phosphine (TCEP) were obtained from Sigma–Aldrich. All other chemicals were purchased from Sigma–Aldrich (USA) and were reagent grade.

Non-radioactive cross-coupling reaction mixtures

Coniferyl alcohol(1), Tyr, Cys, Ser, and Thr were dissolved in 0.01 M sodium phosphate, pH 7.5.

Acknowledgements

This research was supported in part by a research grant from the Department of Energy DE-FG02-07ER15891 and also in part from the Center for LignoCellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001090 (Center funds provided partial support for both F.C. and B.G.D.). The Penn State Proteomics and Mass Spectrometry Core Facility performed the LC/MS analysis.

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Covalent bond formation between amino acids and lignin: Cross-coupling between proteins and lignin

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Amino acid/coniferyl alcohol(1) cross-coupling

Discussion

Conclusions

Chemicals

Non-radioactive cross-coupling reaction mixtures

Acknowledgements

Polym. Degrad. Stab.

Cell

Anal. Biochem.

Trends Plant Sci.

J. Biol. Chem.

Phytochemistry

Phytochemistry

Carbohydr. Res.

J. Biol. Chem.

Phytochemistry

Phytochemistry

Lignin biosynthesis

Annu. Rev. Plant Biol.

The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline/proline-rich proteins

Plant Physiol.

The United-States-Department-of-Energy Biofuels Research-Program

Energ. Sources

Plant cell wall proteins

Annu. Rev. Plant Phys.

Restructuring the cystalline cellulose hydrogen bond network enhances its depolymerization rate

J. Am. Chem. Soc.

Biosynthesis and constitution of lignin

Nature