Membrane-Bound Class III Peroxidases: Unexpected Enzymes with Exciting Functions

Abstract

Class III peroxidases are heme-containing proteins of the secretory pathway with a high redundance and versatile functions. Many soluble peroxidases have been characterized in great detail, whereas only a few studies exist on membrane-bound isoenzymes. Membrane localization of class III peroxidases has been demonstrated for tonoplast, plasma membrane and detergent resistant membrane fractions of different plant species. In silico analysis revealed transmembrane domains for about half of the class III peroxidases that are encoded by the maize (Zea mays) genome. Similar results have been found for other species like thale-cress (Arabidopsis thaliana), barrel medic (Medicago truncatula) and rice (Oryza sativa). Besides this, soluble peroxidases interact with tonoplast and plasma membranes by protein⁻protein interaction. The topology, spatiotemporal organization, molecular and biological functions of membrane-bound class III peroxidases are discussed. Besides a function in membrane protection and/or membrane repair, additional functions have been supported by experimental data and phylogenetics.

Keywords: Arabidopsis thaliana; Class III peroxidase; Medicago truncatula; Oryza sativa; Zea mays; microdomains; phylogenetics; plasma membrane; protein–protein interaction; tonoplast.

Figure 1

Active sites of horseradish peroxidase (HRP) and OsPrx95 and multiple sequence alignment of class III peroxidases. Superposition of active sites of HRP (blue) and OsPrx95 (yellow) was prepared by Phyton-enhanced molecular graphic tool (PyMOL): (a) site view, (b) top view; in HRP Arg-38 and His-42 build the distal active-site structure [39]. The heme group (grey), with δ-meso edge (red), is fixed by His-170 in the active site. (c) Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used for multiple sequence alignment of HRP [40], membrane-bound peroxidases and soluble peroxidases that were used as templates for modelling of structures shown in Figure 2 [36,41,42,43]. Active sites (yellow), calcium binding-sites (grey), conserved cysteine residues for formation of disulfide bridges (blue), transmembrane domains (bold and underlined), signal peptides (italic).

Figure 2

Putative tertiary structures of PM-bound class III peroxidases. Structures were predicted by SWISS-MODEL for (a) ZmPrx01 (3hdlA, 1 TMH, root PM), (b) OsPrx95 (3hdl1A, 3 TMH, root PM), (c) AtPrx64 (3hdl1A, 0 TMH, PM), (d) AtPrx47 (5twt, 1 TMH, PM), and (e) MtPrx02 (5twt1A, 1 TMH, root tip DRM). N-terminal amino acid sequences containing transmembrane domains (marine) were modeled by ab initio with Phyre2 in intensive mode [49]. Ligand binding-sites were predicted by 3DLigandSite [50]. The hypothetical models were prepared by PyMOL (http://pymol.org/) and present the structural characteristics of class III peroxidases. Colors indicate secondary structures: α-helix (red), β-sheets (yellow), loops (green), disulfides (blue), and haem group (grey).

Figure 3

Membrane interaction and spatiotemporal organization of CroPrx01, ZmPrx01 and AtPrx64. (a) Soluble CroPrx01 interacts via an arabinogalactan protein (AGP, blue) with the inner surface of the tonoplast [35,67]. For α-3′,4′-anhydrovinblastine (AVLB) synthesis, the complex anchored next to a proton driven alkaloid antiporter (yellow ellipse) [68,69], (b) ZmPrx01 is anchored in the PM by a transmembrane-spanning domain with its active site at the out-side [22]. For cell wall modification, the enzyme co-localized with Rboh (ruby colored) in microdomains. Simultaneously, superoxide dismutase (SOD, green) and ZmPrx01 regulate ROS levels at the PM [14,70]. (c) Soluble AtPrx64 interact with a transmembrane Casparian strip protein (CASP1, orange) at the PM [28,29,71,72]. A dirigent-like protein (EBS1, grey) facilitates co-localization of the complex with RbohF (ruby colored) in sclerenchyma cells [72]. Casparian strip formation is regulated by MYB36 [29]. For peroxidases solvent accessible surface charges are shown: negative (red); neutral (white), positive (blue).

Figure 4

Phylogenetic analysis of peroxidases from Table 1 and MpPrx92 by the maximum likelihood method. The evolutionary history was inferred by using the maximum likelihood method based on the Jones, Taylor, Thornton (JTT) matrix-based model [110]. The tree with the highest log likelihood (-58984.24) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 110 amino acid sequences. There were a total of 841 positions in the final dataset. Evolutionary analyses were conducted by Molecular Evolutionary Genetics Analysis (MEGA7) [111]. ●, ER; ●, PM; ●, Vac; ■ Golgi; ▲, MoM; ▼, MiM.