Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification

Abstract

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.

Figure 1. Absorption spectra of purified recombinant AtPrx2, AtPrx25, AtPrx53, and AtPrx71.

Spectra from 250 to 800 nm of 10 µM proteins measured by UV-visible spectrometry and inset shows CBB staining of purified proteins (400 ng) after SDS–PAGE (12% gel) with molecular mass markers given in kDa on left.

Figure 2. Spectrophotometric demonstration of Cc ²⁺ oxidation.

A: Spectral changes of Cc ²⁺ during incubation with 0.1 µg rCWPO-C. Optical spectra between 450 and 600 nm recorded for Cc ²⁺ in 50 mM Tris-HCl before (trace a) and after incubation with 0.1 mM H₂O₂ and 0.1 µM rCWPO-C for 180 sec (trace b) and 300 sec (trace c). B–F: Time course of Cc ²⁺ oxidation by recombinant peroxidases. Absorbance monitoring started immediately after H₂O₂ addition; spontaneous Cc ²⁺ oxidation monitored without H₂O₂ addition (trace d); protein concentrations: trace e, 0 µM; trace f, 0.05 µM; trace g, 0.1 µM; trace h, 0.2 µM; trace i, 0.3 µM; and trace j, 0.4 µM; complementary trace (H.D), absorption change with heat-denatured protein at given concentration.

Figure 3. Primary and predicted 3-D structure of AtPrx2, AtPrx25, and AtPrx71.

A: Amino acid alignment of plant peroxidases, HRP-C, AtPrx-53, 2, 25, 71, and ZePrx; conserved Pro139 in AtPrx53 in green structural motifs common to S peroxidases previously proposed (Ros Barceló et al. 2007, Novo-Uzal et al. 2013) in blue; tyrosine and tryptophan residues, presented in three peroxidases, AtPrx-2, 25, and 71, but not in AtPrx53 and HRP-C highlighted in yellow; possible oxidation sites enclosed in box. B: Predicted 3-D structures of AtPrx-2, 25, and 71 by homology-modeling performed with SWISS-MODEL using PDB entry (http://www.pdb.org/pdb/home/home.do) 1FHF (for AtPrx2) and 1PA2 structure (for AtPrx-25 and 71) as template; N and C-terminus of protein molecules labeled; and possible oxidation sites on protein surface in red.

Figure 4. A cobweb chart illustration of the relative oxidation activities of recombinant peroxidases and native HRP-C.

Peroxidase activities listed in Table 3 converted to relative values calculated with the highest activity among six peroxidases set to 1.0; Cc ²⁺ oxidation activity estimated by decreased Cc ²⁺ concentration listed in Figure 3, except for HRP-C; and Cc ²⁺ oxidation by HRP-C previously reported (Sasaki et al. 2004).