Comparative Insilico Analysis of Ascorbate Peroxidase Protein Sequences

Aerobic life has developed by exploiting the abundance of environmental oxygen (O2) in the atmosphere to oxidize organic compounds, thus obtaining chemical energy in a highly efficient manner. Paradoxically, the univalent reduction of molecular oxygen in metabolic reactions produces a plethora of partially reduced intermediates, commonly known as reactive oxygen species (ROS). If their levels are not tightly controlled, these chemical species can react with the majority of biological molecules and cause serious cellular damages [1-2]. ROS are byproducts of aerobic metabolism and are produced in excess within plant cells under abiotic and biotic stresses [3-4]. However, ROS are also important in many physiological processes and their balance is of the utmost importance. As a result, a complex system, comprising enzymatic and nonenzymatic mechanisms, maintains the delicate balance between oxidant and antioxidant compounds in the cell [5]. Ascorbate peroxidase (APX) is known play the most essential role in scavenging ROS and protecting cells against these toxic effects in higher plants, algae, euglena and other organisms [6,9]. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H2O2 to H2O2 using ascorbate as the specific electron donor in this enzymatic reaction [9]. APX is the largest class of the nonanimal peroxidase superfamily, and its members are found in all living organisms except Diplomonads, Parabasalids, Apicomplexa, Amoebozoa, and animals [10]. Increased activity of different APX isoforms in response to environmental stresses such as salinity and drought has been reported in different plant species, indicating possible functional specialization of the respective isoenzymes in eliminating H2O2 in cells [11-12]. APX in higher plants are encoded by small multigene families and different isoforms are classified according to their subcellular localization. Soluble isoforms are found in cytosol and chloroplast stroma, while membrane-bound isoforms are found in peroxisomes and chloroplast thylakoids. The final subcellular localization of the isozyme is determined by the presence of organelle specific targeting peptides and transmembrane domains that are found in the protein N-terminal and C-terminal [13], APXs purified from different plant species and tissues, such as tea leaves, maize (Zea mays) seedlings and leaves, and potato (Solanum tuberosum) tubers, have been isolated in both monomeric and dimeric forms [14]. Expression of this gene has been reported to be enhanced in plants by drought and salt [15-16].


Introduction
Aerobic life has developed by exploiting the abundance of environmental oxygen (O 2 ) in the atmosphere to oxidize organic compounds, thus obtaining chemical energy in a highly efficient manner. Paradoxically, the univalent reduction of molecular oxygen in metabolic reactions produces a plethora of partially reduced intermediates, commonly known as reactive oxygen species (ROS). If their levels are not tightly controlled, these chemical species can react with the majority of biological molecules and cause serious cellular damages [1][2]. ROS are byproducts of aerobic metabolism and are produced in excess within plant cells under abiotic and biotic stresses [3][4]. However, ROS are also important in many physiological processes and their balance is of the utmost importance. As a result, a complex system, comprising enzymatic and nonenzymatic mechanisms, maintains the delicate balance between oxidant and antioxidant compounds in the cell [5]. Ascorbate peroxidase (APX) is known play the most essential role in scavenging ROS and protecting cells against these toxic effects in higher plants, algae, euglena and other organisms [6,9]. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H 2 O 2 to H 2 O 2 using ascorbate as the specific electron donor in this enzymatic reaction [9]. APX is the largest class of the nonanimal peroxidase superfamily, and its members are found in all living organisms except Diplomonads, Parabasalids, Apicomplexa, Amoebozoa, and animals [10]. Increased activity of different APX isoforms in response to environmental stresses such as salinity and drought has been reported in different plant species, indicating possible functional specialization of the respective isoenzymes in eliminating H 2 O 2 in cells [11][12]. APX in higher plants are encoded by small multigene families and different isoforms are classified according to their subcellular localization. Soluble isoforms are found in cytosol and chloroplast stroma, while membrane-bound isoforms are found in peroxisomes and chloroplast thylakoids. The final subcellular localization of the isozyme is determined by the presence of organelle specific targeting peptides and transmembrane domains that are found in the protein N-terminal and C-terminal [13], APXs purified from different plant species and tissues, such as tea leaves, maize (Zea mays) seedlings and leaves, and potato (Solanum tuberosum) tubers, have been isolated in both monomeric and dimeric forms [14]. Expression of this gene has been reported to be enhanced in plants by drought and salt [15][16]. This paper reports in silico characterization of amino acid sequence of heme binding peroxidase from different plants for homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis using various bioinformatics tools proposing new strategies for plant and crop improvement to combat stressful condition.

Retrieval of ascorbate peroxidase protein sequences
For the identification of APX in various plants, the homology search *Corresponding author: Yogesh Kumar Negi, SBS P.G. Institute of Biomedical Science and Research, Balawala-248161 Dehradun, India, E-mail: yknegi@ rediffmail.com, plantstress@gmail.com of the APX proteins was done through Blast search tool of NCBI (http:// www.ncbi.nlm.nih.gov/BLAST/) using Blastp and tblastn algorithm and their amino acid sequence of different source organism available in GenBank were downloaded from NCBI (http://www.ncbi.nlm.nih. gov/). Only reference sequences were retrieved while non reference sequences were removed.

Multiple sequence alignment
All the sequences of APX were aligned using ClustalW [18] to find out the similarity present among the sequences of the same family.

Phylogenetic analysis
Phylogenetic analysis of the sequences was done by Molecular Evolutionary Genetic Analysis (MEGA) software (version 4.0.02) [19], using UPGMA method. Each node was tested using the bootstrap approach by taking 1,000 replications and a random seeding of 64,238 to ascertain the reliability of nodes. The number is indicated in percentages against each node. The branch lengths were drawn to scale indicated.

Motif analysis
Analysis of conserved motifs was performed by means of the online MEME (Multiple Expectation Maximization for Motif Elicitation) tool version 3.5.7 [20] using minimum and maximum motif width of 20 and 50 residues respectively and maximum number of 10 motifs, keeping rest of the parameters at default.

Multiple sequence alignment
A total of 64 full-length amino acid sequences of Ascorbate peroxidase (APX) enzyme from different plants were considered for comparative In Silico analysis (Table 1).
To investigate the APX sequence features among various plants we performed multiple sequence alignments of the 64 amino acid sequences of APX. Conserved region of all proteins are shown in Figure  1 (shown as suppelmentary). Multiple sequence alignment highlighted the sequence conservation of amino acid residues among different members of APX families in the species. This conservation however, is concomitant with differences sufficient enough to support variations which are subsequently reflected at the structural and functional levels.

Phylogenetic analysis
To examine the phylogenetic relationship among APX from different plants a rooted tree was constructed from alignments of their amino acid sequences (Figure 2). The phylogenetic analysis of APX across all plant species clearly reveals four clusters: cluster A, cluster B, cluster S.No. Plant Total No.

Motif analysis
An extensive search of the motifs and their positions was done by MEME software which identified several conserved motifs in the protein sequences of APX (Table 2 and Figure 3). Motif analysis also communicated the same fundamental necessity for the development of this gene family. Motifs which contain the signature sequences are either well conserved or are having substitutions which do not change their activity, while the ones which do not have a direct impact on the active site contain altered residues and are clearly the outcome of accumulation of mutations or have been subjected to rearrangements. A total of ten motifs labelled as 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 were observed in all 64 sequences when subjected to MEME [21][22][23]. In all plant heme peroxidase, motif-1 was most commonly observed which is functionally related to its detoxication of H 2 O 2 or reactive oxygen species both cytosolic and chloroplast cell compartment as well as having heme binding peroxidase properties. While Motif-4, which also have similar function as Motif-1 was present in all APX isoforms. Motif 2 contains Casein kinase II phosphorylation site and signature of chloroplastic and cytosolic ascorbate peroxidase [24]. Beside this, Motif-3 & 5, 7, and 9 also most frequently present in APX isoforms which are functionally releted with chloroplastic and cytosolic and non animal peroxidase [25]. Motif 6 is present in all APX isoforms  Table 3.

Conclusion
In silico analysis of ascorbate peroxidase protein sequences and its comparison with other APX has revealed the sequence-based similarity existed among different APX isoforms and clustering in distinct groups based on its source among different plants and nature of the mechanism of enzymatic activity against the antioxidant defense mechanism in plants. In silico domain analysis confirms the existence of the different groups of ascorbate peroxidase based on the presence of unique domains, a heme binding domain found in all isoforms of APX. The presence or absence of specific domains was directly in relation with the structural and functional organization of different isoforms of ascorbate peroxidase.
Amino acid sequence similarity specific for different groups could be utilized for designing strategy for cloning the putative genes based on PCR amplification using degenerate primers and potentially useful for the development of transgenic crop plants tolerant to abiotic stresses.