Prediction of some peroxidase functions in Arabidopsis thaliana L. by bioinformatic search

Peroxidases of class III are common in various organisms. They are involved in lignin biosynthesis and plant protection against stressors. Peroxidases are presented in many isoforms, whose role is not always clear. The aim of this study is to analyze the amino acid sequences of reference peroxidases with known functions and peroxidases from Arabidopsis thaliana L. whose functions are unknown and to consider their putative roles in lignin biosynthesis. The structural and functional organization of peroxidases was analyzed by bioinformatical methods applied to open Internet sources. Seven reference peroxidases were chosen from four plant species: Zinnia sp., Armoracia rusticana P.G. Gaertn., Lycopersicon esculentum L. и Populus alba L. Twenty-four amino acid sequences of homologous peroxidases from A. thaliana were selected for the analyses with the BLAST service. Their molecular weights and isoelectric points were calculated. Multiple alignments of amino acid sequences and phylogenetic analysis were done. Sites of binding to monolignol substrates were identified in seven peroxidases from A. thaliana , and the enzymes were assigned to the groups of S-or G-peroxidases. Amino acid replacements in the primary structures of peroxidases were analyzed. Peroxidases from A. thaliana were clustered with reference peroxidases. They formed six clusters on the phylogenetic tree, three of which contained only A. thaliana peroxidases. Peroxidases within each cluster had similar molecular weights and isoelectric points, common localization of expression, and similar functions. Thus, the use of bioinformatics, databases, and published data bring us to assumptions as to the functions of several A. thaliana class III peroxidases. AtPrx39 peroxidase was shown to be affine to sinapyl alcohol; AtPrx54, to p -coumaryl and coniferyl alcohols. They are likely to participate in lignin biosynthesis.


Introduction
Peroxidases are the group of enzymes that catalyze the oxidation of a substrate with the presence of hydrogen peroxide.The superfamily of "plant" peroxidases (those of plants, fungi, and bacteria) is divided into three classes based on their structural and catalytic properties.All peroxidases contain 10 homologous α-helixes.Class I and class II have one specific α-helix, and class III peroxidases have three specific α-helixes (Hiraga et al., 2001).
Living organisms contain many peroxidase isoforms, and their amino acid sequences are similar by less than 20 %.A high level of conservation characterizes five amino acid positions essential for the folding of α-helixes, assembly of subunits, and catalytic properties of the enzymes (Hiraga et al., 2001).
Higher plants contain class I and III peroxidases, which differ in structure, function, and location in the plant cell.Ascorbate peroxidase (EC 1.11.1.11)and glutathione peroxidase (EC 1.11.1.9)belong to class I.They are located in chloroplasts, peroxisomes, and cytoplasm.Class I peroxidases are distinguished by high specificity to an oxidizable substrate.Class III peroxidases (EC 1.11.1.7)include enzymes that are located in vacuoles and secreted into the apoplast.They oxidize various substrates.Class III plant peroxidases are encoded by a large family of genes: 73 have been identified in Arabidopsis thaliana L. and 138 in Oryza sativa L. (Welinder et al., 2002;Passardi et al., 2004a).Class III peroxidases act as components of the antioxidant system of plants and, at the same time, can form reactive oxygen species (Passardi et al., 2004a).The dual functions of peroxidases allow them to take part in many physiological processes: protection against pathogens (Passardi et al., 2004b), wound healing, auxin and anthocyanin catabolism, and porphyrin metabolism (Cosio, Dunand, 2009;Jovanovic et al., 2018).
Apoplastic peroxidases are involved in the biosynthesis of cell wall components, such as lignin and suberin.Lignin is an aromatic phenolic heteropolymer with a disordered structure, covalently associated with polysaccharides of the secondary cell wall and responsible for its strength and hydrophobicity.The composition and amount of lignin in the cell wall change in the course of plant ontogenesis (Boerjan et al., 2003) and in response to different stress factors (Liu et al., 2018).
Despite the large amount of research focusing on class III peroxidases, only few isoforms have been shown to participate in lignin biosynthesis in herbaceous (Zinnia sp., Armoracia rusticana P.G.Gaertn., Lycopersicon esculentum L.) and woody (Populus alba L.) plants (Quiroga et al., 2000;Aoyama et al., 2002;Sasaki et al., 2004;Sato et al., 2006;Marjamaa et al., 2009).Class III peroxidases can oxidize three monolignols; however, most isoforms oxidize coniferyl and p-coumaryl alcohols and only few of them use sinapyl alcohol as a substrate in vitro (Barcelo et al., 2007).
The structures of peroxidases ZePrx34, ZPO-C, CWPO-C, HRP, HRP-A2A, HRP-C1C, and TPX1, which can be considered reference ones, have been studied in detail (Quiroga et al., 2000;Aoyama et al., 2002;Sasaki et al., 2004;Gabaldon et al., 2005;Sato et al., 2006).The attention to peroxidases is due to their function in the formation of plant resistance to oxidative stress caused by both abiotic and biotic factors, as well as to their participation in lignin biosynthesis and plant growth.Bioinformatic analysis of peroxidases with unknown functions is of fundamental (determination of the enzyme functions) and practical (design of genetic constructs to create resistant plants or plants with a modified cell wall) significance.The purpose of this work is to analyze the functions of A. thaliana peroxidases based on the similarity to the amino acid sequences of reference plant peroxidases for which the involvement in cell wall lignification is known.
The phylogenetic tree based on A. thaliana peroxidase proteins was built by the Neighbor-Joining method (Sanou, Nei, 1981)  computed by the p-distance method (Nei, Kumar, 2000).The bootstrap test included 1000 replicates, and the results are shown nearby the branches (Kumar et al., 2016).
Information about the expression of A. thaliana peroxidase genes at different stages of development was obtained from the bio-array resource for plant functional genomics (http://bar.utoronto.ca).Analysis of peroxidase functions was carried out with regard to information stored in the Arabidopsis Information Resource (www.arabidopsis.org)by gene identifiers in TAIR.We considered information from the Annotations, GO Biological Process section on the involvement of peroxidases in stress reactions, growth, and lignification of the cell wall.Amino acid sequence alignments were built with the CLUSTAL algorithm for multiple sequence alignment in MUSCLE 3.8 (https://www.ebi.ac.uk/Tools/msa/).Highly conservative and semiconservative domains, structural motifs were identified.
The isoelectric points (pIs) and molecular weights of A. thaliana peroxidases differ from those of reference enzymes.In particular, the pI value of peroxidase AtPrx36 is in the more acidic pH range compared to ZePrx34.70, and the protein has a higher molecular weight (38.24 vs. 34.24kDa, respectively).AtPrx13 peroxidase is characterized by an acidic pI value (4.74), whereas pI for HRP is 8.35.AtPrx32, 37 and 23 peroxidases have pIs within 6.62-7.97,and their molecular weights vary from 38.10 to 38.85 kDa, whereas the pI and molecular weight of HRP_A2A protein are 4.62 and 35.03 kDa, respectively.
It is seen that A. thaliana peroxidases differ in pI values and molecular weights from reference enzymes and they are expected to differ in their affinity to the substrate and in functions.It is known that basic peroxidases (isoelectric point > 7.0) can oxidizing p-coumaryl, coniferyl and sinapyl alcohols (Kukavica et al., 2012), while acidic peroxidases (isoelectric point < 7.0) are poorly capable of oxidizing sinapyl alcohol (Barcelo et al., 2004).Therefore, the roles of basic and acidic peroxidases in cell wall lignification may be different.Plant peroxidases with high ability to oxidize coniferyl alcohol (CWPO-A, HRP-C1C and AtPrx53) or sinapyl alcohol (CWPO-C from P. alba, ZePrx from Z. elegans, AtPrx4) are described.
The phylogenetic tree of these peroxidases shown in Figure is constructed by amino acid alignment.The reference peroxidases and A. thaliana enzymes form six clusters.The first one includes HPR-C1C, AtPrx33, 34, and 32 peroxidases with a high bootstrap support value of 72-100 %.The second cluster groups peroxidases, homologous to HRP_A2A: AtPrx2 and 54 (bootstrap support 100 %).AtPrx52 and 4 peroxidases, homologous to ZePrx34.70, form the third cluster with bootstrap support of 98-99 %.AtPrx47, 64, and 66 peroxidases group in the fourth cluster together with ZPO-C peroxidase (92-100 % bootstrap support).The fifth cluster on the phylo-genetic tree combines peroxidases TPX1 and AtPrx3 and 39 with a bootstrap support of 100 %.The sixth cluster consists of HRP, CWPO-С, AtPrx71, 62, and 69 peroxidases with bootstrap support value of 72-100 %.
With materials from the BAR and TAIR databases, the peroxidase functions and expression sites were identified for the enzymes of clusters 1-6 (Table 2).The Table 2 does not include clusters A, B, or C, formed by homologous proteins of A. thaliana.
Functions of some A. thaliana peroxidases have been studied in mutants with knocked-out genes and transgenic plants.
According to experimental studies, peroxidase HRP-C1C from A. rusticana most effectively oxidizes coniferyl alcohol in vitro (Sasaki et al., 2004).The most homologous HRP-C1C peroxidases AtPrx33 and AtPrx34 are involved in root growth and cell elongation (Irshad et al., 2008) and in an oxidative burst, when pathogens penetrate into the cell (Bindschedler et al., 2006).AtPrx32 peroxidase is involved in cell elongation (Irshad et al., 2008).Thus, there is no data on the participation of cluster 1 enzymes in cell wall lignification.
Purified peroxidase HRP_A2A from A. rusticana efficiently oxidizes guaiacol in vitro (Krainer et al., 2014).According to the BAR database, the AtPRX2 and AtPRX54 genes are expressed in its seedling roots and hypocotyl and in the roots of juvenile plants (see Table 2).Mutants of A. thaliana atprx2 are characterized by a reduced total lignin content, changes in lignin composition, and plant biomass decrease (Shigeto et al., 2013).
The isoform ZePrx34.70 from Z. elegans, catalyzing the oxidation of sinapyl alcohol, is expressed in roots and hypocotyl and involved in lignification (Gabaldon et al., 2005).Among the analyzed peroxidases, AtPrx4 and AtPrx52 from A. thaliana are homologous to ZePrx34.70, as confirmed in (Herrero et al., 2013a).According to (Fernandez-Pereza et al., 2015), the AtPrx4 gene is expressed in roots, stems, and leaves, and it affects the plant growth on long days.The product of its expression is involved in syringol polymerization.
Purified ZPO-C peroxidase from Z. violacea uses both synapyl and coniferyl alcohol as a substrate in vitro (Sato et al., 2006).Homologous AtPrx66 takes part in cell wall lignification of forming vessels (Sato et al., 2006).It was shown that the homolog AtPrx64 also plays a role in xylem lignification (Yokoyama, Nishitani, 2006).
The gene for the basic peroxidase TPX1 of L. esculentum is specifically expressed in root xylem and involved in lignification and suberization (Quiroga et al., 2000).An increase in lignin content has been shown in transgenic L. esculentum plants with overexpression of TPX1 (Mansouri et al., 1999).Homologous peroxidase AtPrx3 is involved in lignification (see Table 2).AtPrx3 cationic peroxidase transcripts were found in the seedlings and roots, and their participation in the response of plants to salt stress and drought was shown (Llorente et al., 2002).The role of AtPrx39 peroxidase in cell wall lignification has not been studied.However, the AtPRX39 gene is expressed in the root transport zone.It affects the development of the root system (Tsukagoshi et al., 2010).Peroxidase CWPO-C from P. alba is a cationic isoform of the enzyme efficiently polymerizing sinapyl alcohol in vitro (Aoyama et al., 2002).HRP is a cationic isoform with high ability to oxidize coniferyl alcohol.Peroxidase isoenzymes, such as HRP and CWPO-C, have been shown to catalyze single-electron oxidation of sinapyl alcohol using coniferyl alcohol as a radical mediator (Aoyama et al., 2002).AtPrx71 peroxidase is the closest homolog of HRP.It participates in the formation of secondary xylem (Yokoyama, Nishitani, 2006) and in the response to biotic factors (Chassot et al., 2007).AtPrx62 peroxidase expression increases in response to heavy metal ions and plant pathogens (Cosio, Dunand, 2009).
The genes encoding peroxidase AtPrx32 and 37 are expressed in the root and hypocotyl and involved in lignification (see Table 2).Overexpression of AtPRX37 in transgenic A. thaliana causes a decrease in plant growth rate, affects the development of xylem, and ultimately leads to the formation of a dwarf phenotype.Presumably, AtPrx37 peroxidase is involved in the regulation of plant growth through the cell wall lignification process (Pedreira et al., 2011).AtPrx72 and 36 peroxidases differ in localization in plant tissues and their functions.The AtPRX72 gene is expressed in roots and stems (Valerio et al., 2004).The AtPRX36 gene is expressed in the hypocotyl, where it takes part in cell elongation (Irshad et al., 2008); in the endosperm; and the seed coat (Kunieda et Prediction of some peroxidase functions in Arabidopsis thaliana L. by bioinformatic search al., 2013).Analysis of A. thaliana mutants for the AtPRX72 gene showed a decrease in lignin content and the number of syringyl units.In addition, the mutants were characterized by slow growth, decrease in stem diameter, and smaller numbers of shoots and leaves (Herrero et al., 2013b).Thus, several peroxidases, clustered in groups 2-6, can oxidize monolignols and participate in the lignification of cell walls.
We anticipate that peroxidases combined into a common cluster on a phylogenetic tree are predominantly expressed in the same plant organs and perform similar functions (see Table 2).Of all peroxidases, the participation of AtPrx54 and 39 in physiological processes is least understood.We infer from the results of data analysis that AtPrx54 peroxidase performs functions similar to AtPrx2: it is involved in lignification, growth, and response to abiotic stress.AtPrx39 peroxidase is involved in lignification and responses to biotic and abiotic stress.Assuming the concept of the evolutionary origin of proteins (Gabaldon, Koonin, 2013), we conjecture the participation of AtPrx54 and 39 peroxidases in cell wall lignification processes, since they are orthologs of HRPA2A and TPX1, respectively.AtPrx69 and 62 peroxidases, combined into a common cluster with AtPrx71 peroxidase, are also highly likely to be involved in cell wall lignification.
The functions of homologous proteins can be determined based on their domain structure and substrate binding sites.Multiple alignment of amino acid sequences was previously performed (data not shown).It included reference and A. thaliana peroxidases combined into a common cluster (clusters 1-6 on the phylogenetic tree, see Figure ).AtPrx54 peroxidase was shown to have structures like AtPrx4 and HRP_A2A; and AtPrx39 peroxidase was highly homologous to TPX1, CWPO-C, and HRP proteins.Highly conservative and semiconservative sections, structural motifs characteristic of the analyzed class III peroxidases were identified (see Suppl. 2, 3).
Thus, peroxidases are distinguished by structural motifs, and, accordingly, affinity to oxidizable substrates.The structural motifs that are necessary and sufficient to polymerize G-monolignols are V78, 95-VSCSD, S98, 105-SEA, F185, and N281.S-Peroxidases have motifs absent from G-peroxidases: I80, 95-VSCAD, A98, 105-ARD, Y178, and K268.The 95-VSCAD motif determines the ability of peroxidases to polymerize both syringaldazine and sinapyl alcohol (Barcelo et al., 2007).In addition, the affinity of peroxidase to the substrate is affected by hydrophobic interactions between the substrate and the enzyme, which involve the amino acids at positions P69, I138, P139, S140, R175, and V178 (Barcelo et al., 2007).Studies of the ATP A2 peroxidase structure from A. thaliana have shown that hydrophobic interactions between the sinapyl alcohol and the amino acid residues in position I138 and P139 do not allow the enzyme to use sinapyl alcohol as a substrate (Ostergaard et al., 2000).
The G-peroxidases whose amino acid sequences were analyzed include AtPrx2 and 54, HRP_A2A.They are characterized by structural motifs V78, 95-VSCSD, S98, 105-SEA, F186, and N281.The hydropathicity of the site of substrate binding is determined by amino acid substitutions increasing hydrophobicity: proline to alanine at position 96, isoleucine to leucine at position 138, and isoleucine to The peroxidase protein phylogenetic tree from A. thaliana based on sequence alignments of the encoded protein (Sanou, Nei, 1981).
The evolutionary history was reconstructed by the Neighbor-Joining method.The optimal tree with the sum of branch length 5.06962574 is shown.The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown nearby the branches.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to construct the phylogenetic tree.The evolutionary distances were computed by the p-distance method (Nei, Kumar, 2000).They are presented as numbers of amino acid differences per site.The analysis involves 31 amino acid sequences.All positions containing gaps and missing data are eliminated.A total of 268 positions are present in the final dataset.Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).For protein database NCBI accession numbers of the sequences used for the building of phylogenetic tree see phenylalanine at position 142.Replacements at sites with hydrophilic amino acids (glycine to proline at position 68, isoleucine to leucine at position 138, arginine to glutamine at position 175, glycine to valine at position 177, or valine to threonine at position 178) do not change the properties of the substrate-binding sites.The 138-IPS hydrophobic motif determines the conformation of the protein and the hydrophobicity of the substrate-binding site.Thus, AtPrx54 peroxidase has sites that enable it to polymerize p-coumaryl and coniferyl alcohols.

Conclusion
Plant peroxidases of class III from different plant families are similar to each other in amino acid sequences, tissue localization, and functions.Structure-functional regions were identified in the peroxidases on the base of amino acid sequence homology.These regions allow inferences as to the substrate specificity of the peroxidases.The results show that AtPrx39 oxidizes sinapyl alcohol and belongs to S-peroxidases; AtPrx54 oxidizes p-coumaryl and coniferyl alcohols and belongs to G-peroxidases.Therefore, AtPrx39 and 54 peroxidases can participate in the polymerization of monolignols in lignin biosynthesis.Thus, the use of bioinformatic methods in the MEGA 7 program based on sequence alignments of the encoded protein.The evolutionary distances were 617 биоинформатика и клеточная биология / bioinformatics and cell biology

Table 1 .
Prediction of some peroxidase functions in Arabidopsis thaliana L. by bioinformatic search Annotation of class III peroxidases from A. thaliana with high levels of similarity (Score and E-value) to reference peroxidases.The TAIR acc.no., NCBI acc.no., molecular weight, and isoelectric point are indicated for each protein

Table 2 .
The function of class III peroxidases from A. thaliana : The data about peroxidases functions were obtained from the Arabidopsis Information Resource (www.arabidopsis.org).Localization of expression on different stages of development was analyzed using the bio-array resource for plant functional genomics (http://bar.utoronto.ca). Note