19 IJBMRF20141667 Yeasmeen Ali - Semantic Scholar

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Original Article

In silico Analysis of Chloroperoxidase and Lignin Peroxidase of Pathogenic Fungus Macrophomina phaseolina a

b

a

b

Yeasmeen Ali , Mohd Omar Faruk Sikder , Lolo Wal Marzan , Farjana Sharmen b, Md Mursalin Khan , a Md Amzad Hosain , * a

Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong- 4331.

b

Bioinformatician, Basic and Applied Research on Jute Project, Bangladesh Jute Research Institute, Dhaka- 1207.

ARTICLE INFO

ABSTRACT

Keywords:

Macrophomina phaseolina is a destructive fungus that affects more than 500 plant species throughout the world. Lignin degradation of the host plant is crucial step for the pathogenesis of this fungus. Chloroperoxidase and lignin peroxidase are two important enzymes for lignin degradation. To develop an effective antagonist against this fungus, understanding these proteins is necessary. In this study, we have reported physico-chemical characteristics, phylogenic relationship, secondary structure, 3-D structure, motifs of 7 chloroperoxidase and 3 lignin peroxidase proteins. Moreover, pockets as well as conserved residues in the motif sequence were identified as the target for site-directed mutagenesis of chloroperoxidase and lignin peroxidase. This mutagenesis or designing target ligands against these proteins may stop the ligninolytic activity of M. phaseolina, which would save economically important plants.

Motif Botryosphaeriaceae Physicochemical property Active site Secondary structure.

c Copyright 2010 BioMedSciDirect Publications IJBMR -ISSN: 0976:6685. All rights reserved.

1. Introduction Macrophomina phaseolina is a Botryosphaeriaceae fungus that belongs to phylum ascomycota. It is one of the most devastating soil and seed borne pathogens infecting more than 500 plant species [1]. The plant species includes jute [2], cotton [3], maize [4], pulses [5], sunflower [6] etc. This fungus under favorable environmental conditions can cause diseases like charcoal rot, stem rot, damping off, seedling blight, collar rot, and root rot in various important crops [7]. Disease development is considered to be associated with several factors such as heat stress, soil water deficit, physiological stress or coarse soil texture [8]. Due to having sclerotia [thickwalled resistant hyphal mat] in soil and plant debris, it is difficult to control M. phaseolina [9]. This pathogen is widely distributed throughout the world especially in tropical and subtropical countries [10]. In Bangladesh, due to pathogenicity of this fungus only jute fiber production rate is reduced by 30% [11].

* Corresponding Author : Yeasmeen Ali Lecturer, Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong- 4331. E-mail: [email protected]

c Copyright

2010 BioMedSciDirect Publications. All rights reserved.

In 2012, the genome sequence as well as gene prediction of M. phaseolina was accomplished. The genome size was about 49 Mb and about 14,249 protein-coding genes were predicted [11]. It possesses ligninolytic activity, which is the result of combination of different enzymatic system such as slaccases, lignin peroxidases, galactose oxidases, chloroperoxidases, haloperoxidases, and heme peroxidases. Lignin peroxidase [Lip] is an extracellur enzyme [12], which with the help of a cofactor, Veratryl alcohol interacts with lignin polymer. Likewise, chloroperoxidase [CPO] is also an extraxellur enzyme that requires heme or vanadium as a cofactor [13]. This enzyme acts on lignin by oxidization of Cl− to hypochlorous acid [HOCl] or a similarly reactive chlorine electrophile[14]. However, the mechanism is not still well understood. Our study is to analyze the chloroperoxidase and lignin peroxidase of M. phaseolina, which will help to understand the pathogenesis of this devastating fungus. We have analyzed 7 chloroperoxidase and 3 lignin peroxidase enzymes and found different important investigations such as physic-chemical characteristics, functional motifs, 3D structure of these proteins. We also showed the active site pockets, different amino acids that could be structurally and functionally critical as well as

Yeasmeen Ali et.al Int J Biol Med Res. 2014; 5(3): 4246-4257 4247

phylogenetic relationship among these peroxidases. All of these findings would decipher an effective way to block the activity of chloroperoxidase and lignin peroxidase of M. phaseolina to protect different economically important crop species. MATERIALS AND METHODS: Retrieval of Macrophomina phaseolina chloroperoxidase and lignin peroxidase protein sequences: Protein sequences of M. phaseolina were retrieved from NCBI protein database in FASTA format. 7 chloroperoxidase and 3 lignin peroxidase protein sequences were selected for analysis. [http://www.ncbi.nlm.nih.gov/]. Physico-chemical characterization: Different properties including number of amino acids, molecular weight, theoretical isoelectric point [pI], amino acid composition [%], number of positively [Arg + Lys] and negatively charged [Asp + Glu] residues, extinction co-efficient, instability index, aliphatic index and Grand Average of Hydropathicity [GRAVY] were calculated using ExPASy's ProtParam tool [15] [http://expasy.org/tools/protparam.html]. The crystallization tendency of the proteins was determined by using the CRYSTALP2 W E B S E RV E R [ 1 6 ] [ h t t p : / / b i o m i n e ws.ece.ualberta.ca/CRYSTALP2.html] that accepts protein sequences in the FASTA format. The CRYSTALP2 is a kernel-based method that uses the composition and collocation of amino acids, hydrophobicity and isoelectric point of the given sequences to estimate the crystallization propensity of the proteins. Characterization of secondary structure: Secondary structure prediction was performed by SOPMA [17] [ S e l f - O p t i m i z e d P re d i c t i o n M e t h o d w i t h A l i g n m e n t , http://npsapbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/np sa_sopma.html] tool The input protein sequences were given in FASTA format. The number of conformational states was adjusted to four in order to predict Helix, Sheet, Coil and Turn while other parameters were set as default. By using this software, Alpha helix [Hh], 310 helix, [Gg], Pi helix [Ii], Beta bridge [Bb], Extended strand [Ee], Beta turn [Tt], Bend region [Ss], Random coil [Cc], Ambigous states, and other states were predicted. Protein 3D structure prediction: The 3D structures of chloroperoxidases and lignin peroxidases were predicted using the 3D-JIGSAW [version 3] comparative m o d e l i n g s e r v e r [ 1 8 ] [ h t t p : / / bmm.cancerresearchuk.org/~3djigsaw/], which accepts sequences in FASTA format. This program constructs 3D models for given protein sequences based on the known homologous proteins structure. It uses HMM for searching homologous templates in sequence databases [PFAM+PDB+nr] and splits the query sequence into domains. The good templates with maximum coverage of the queries are then used for modeling and the generated models are represented as PDB file format.

Detection of motif: The motif scan tool [19, 20] [http://myhits.isb-sib.ch/cgibin/motif_scan] was used to identify the motifs and their locations in the sequence of Lignin peroxidase and chloroperoxidase. These protein sequences were given as input data [FASTA format] and scanned against 'PROSITE Patterns'. The selected motifs were then visualized by using Swiss- Pdb Viewer V4.1.0 [21]. Locations, nature, match score and match details of the motifs were analyzed. It was aimed to find out the conserved regions of these motifs. At this point of view, multiple sequence alignment [MSA] of these motifs was performed using EBI Clustal Omega [22]. [https://www.ebi.ac.uk/Tools/msa/clustalo/]. As expected, a number of conserved regions were found and their length, conserved amino acids were analyzed. Active site prediction: Active site analysis was performed using Computed Atlas of Surface Topography of Protein [CASTp]. CASTp for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CASTp identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. Most frequently, the largest pocket/cavity has been the active site, but there are a number of instructive exceptions [23]. Ligand volume and binding site volume are somewhat correlated, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands [24]. Analysis of active sites is important for the modeled protein as a precursor to further work on its docking studies, and to shape the process of making a grid before docking. Prediction of Evolutionary relationship: Evolutionary tree among these 10 lignin degrading protein sequences were aligned using multiple sequence alignment tool C l u s t a l o m e g a [ 2 5 ] [ h t t p s : / / w w w. e b i . a c . u k /Tools/services/web_clustalw2/toolform.ebi] , followed by construction of evolutionary tree using the protein sequences in FASTA format as input data The alignment parameters were tuned finely to find out the best alignment and Neighbor Joining [NJ] method was selected to construct the tree. RESULTS: Physico-chemical characterization: Isoelectric point [pI] is a pH in which net charge of protein is zero. pI of lignin peroxidase 2 and 3 were observed to lie in the acidic range, while the rest of the proteins occur in alkaline range. From the study of instability index, it was found that all peroxidases are stable [except Chloroperoxidase 7], as instability index value less than 40 indicates stability of a protein


[Supplementary file, Table 1]. Additionally, Aliphatic index [AI] refers to the relative volume of a protein occupied by its aliphatic side chains. The higher the Aliphatic index of proteins, the more thermally stable the proteins. Aliphatic index of chloroperoxidase 1 [100.11] and 5 [90.84] classifies them as most thermostable, closely followed by other chloroperoxidases [chloroperoxidase 3, 4, 6 and lignin peroxidase 9]. Grand average of hydropathicity index [GRAVY] indicates the interaction of the proteins in water. Chloroperoxidase 1 [0.099] and lignin peroxidase 1 [0.022] are hydrophobic [due to positive GRAVY values]. GRAVY values of other peroxidases were observed within a wide range of -0.055 to 0.54 [hydrophilic]. The amino acid composition of each peroxidase sequence was calculated by using ExPASY's ProtParam tool [supplementary file, Table 2]. High percentage of alanine [above 9.9] and serine [above 8.5] was found in all three lignin peroxidases compared to other amino acid. In all chloroperoxidase [except chloroperoxidase 7], leucine content was found significant. Again, chloroperoxidase 2, 4 and 5 have a glycine content above 8.9. Moreover, a good percentage of serine was found in chloroperoxidase 3 [9.6] and 7 [9.2] and also chloroperoxidase 7 is proline rich [10.0%]. There are some properties of the proteins such as isoelectric point, hydrophobicity, and the frequency of certain collocated di and tripeptides that are pivotal indicators of crystallization [16]. The CRYSTALP2 accounts all such characteristics of given sequences for estimating the confidence of crystallization.The higher the confidence, the more probable that protein is crystallizable and vice versa. Among the 7 chloroperoxidases and 3 lignin peroxidases, chloroperoxidase 5 showed the highest confidence of crystalizaiton that was 0.61 which was followed by chloroperoxidase 2 [0.565] and chloroperoxidase 6 [0.523], respectively [supplementary file, table 1]. In contrast, lignin peroxidase 3 represented the least confidence of crystallization [0.221]. Each of the the remaining chloroperoxidases exhibited higher confidence of crystallization than that of remaining lignin peroxidases. From the CRYSTALP2 result, it can be assumed that chloroperoxidases are more likely to be crystallized than lignin peroxidases. Among all chloroperoxidase, chloroperoxidase 5 represents maximum propensity for crystallization. Characterization of secondary structure: SOPMA analysis of secondary structure of chloroperoxidase and lignin peroxidase protein sequences results the predominance of random coil which is followed by alpha helix, extended strand, and beta sheet, respectively [supplementary file, table 3]. In case of Chloroperoxidase 1, Lignin Peroxidase 1, it was found that alpha helix exceed the random coil. Protein 3D structure: The 3D structure of protein is very crucial for comprehending the protein functions, their sub-cellular localization as well as protein-protein interactions. Based on homology modeling, 3DJIGSAW results showed five 3D models for each of the given

sequences and ranked them according to the scores of ramachandran plot. For each of the proteins, the top ranked 3D model was selected from the given models [supplementary file, fig 1]. The 3D models generated by 3D JIGSAW were then used further for the motif detection and pocket findings. Motif Identification: Chloroperoxidase and lignin peroxidase are found in M. phaseolina as well as many other fungi, posing a major role in lignin degradation [11, 31]. Motifs for chloroperoxidase and lignin peroxidase were identified using Motif scan tool [Fig 1, Supplementary file, table 4]. If we look into the conserved regions [Fig 2], a number of residues are revealed which are already known to have important role in catalysis. These conserverd regions are predominated by cysteine, proline, aspartate, asparagines, histidine, alanine and glycine. Active site analysis: The active sites of chloroperoxidase and lignin peroxidase were predicted [fig 3]. Further, in this study, we have also reported the best active site area of the experimental enzymes as well as the number of amino acids involved in it; showed the number of pockets, with their area and volume [Supplementary file, fig 2]. In case of the lignin peroxidase, the volume of the pockets is quite similar and average is 2157 Å³. There are some variations in the area of pockets; for lignin peroxidase 2, it is 1100.9 Å2 [Supplementary file, table 5]. On the other hand, for lignin peroxidase 3, it is highest 1857.8 Å2. For lignin peroxidase 1, the area is between the two others-1542.7 Å2. But from the visualization we can see a similar pocket structures. In the analysis of the chloroperoxidases we find similar pocket sizes and structures too. For the chloroperoxidase 2, chloroperoxidase 3, chloroperoxidase 4, chloroperoxidase 5 and chloroperoxidase 6, the average pocket area is 2301.7 Å2 and average pocket volume is 3428.6 Å3. Among these, the highest area showed by chloroperoxidase 4 that is 3072.7 Å2 and volume is 5042 Å3. There are two chloroperoxidases show major variations; those are chloroperoxidase 1 and chloroperoxidase 7. For chloroperoxidase 1, area is 697.6 Å2 and volume is 907.5 Å3. In case of chloroperoxidase 7, we identified paramount variation; it is totally different from other chloroperoxidases regarding the area [208.4 Å2], volume [203.8 Å3] and pocket. From the visualization, we can see a similar pocket structure among the chloroperoxidases other than the chloroperoxidase 7.


Fig 1: Motifs in Chloroperoxidase and Lignin peroxidase. Insets show most common amino acids in motifs.


Fig 2: Conserved regions in chloroperoxidase [top] and lignin peroxidases [bottom] motifs using boxshade.

Active site analysis: The active sites of chloroperoxidase and lignin peroxidase were predicted [fig 3]. Further, in this study, we have also reported the best active site area of the experimental enzymes as well as the number of amino acids involved in it; showed the number of pockets, with their area and volume [Supplementary file, fig 2]. In case of the lignin peroxidase, the volume of the pockets is quite similar and average is 2157 Å³. There are some variations in the area of pockets; for lignin peroxidase 2, it is 1100.9 Å2 [Supplementary file, table 5]. On the other hand, for lignin peroxidase 3, it is highest 1857.8 Å2. For lignin peroxidase 1, the area is between the two others-1542.7 Å2. But from the visualization we can see a similar pocket structures. In the analysis of the chloroperoxidases we find similar pocket sizes and structures too. For the chloroperoxidase 2, chloroperoxidase 3, chloroperoxidase 4, chloroperoxidase 5 and chloroperoxidase 6, the average pocket area is 2301.7 Å2 and average pocket volume is 3428.6 Å3. Among these, the highest area showed by chloroperoxidase 4 that is 3072.7 Å2 and volume is

5042 Å3. There are two chloroperoxidases show major variations; those are chloroperoxidase 1 and chloroperoxidase 7. For chloroperoxidase 1, area is 697.6 Å2 and volume is 907.5 Å3. In case of chloroperoxidase 7, we identified paramount variation; it is totally different from other chloroperoxidases regarding the area [208.4 Å2], volume [203.8 Å3] and pocket. From the visualization, we can see a similar pocket structure among the chloroperoxidases other than the chloroperoxidase 7. Fig 3: Pockets [active site] of the enzymes

Yeasmeen Ali et.al Int J Biol Med Res. 2014; 5(3): 4246-4257 4251 Evolutionary relationship: Phylogenetic tree shows evolutionary relationship among all the ten proteins. There are two major clades on the tree. All chloroperoxidases [except chloroperoxidase 1] reside in a common clade whereas, three lignin peroxidases reside in another tree. Chloroperoxidase 1 seems to be the outgroup of the tree. Fig 4: Phylogenetic relationship among chloroperoxidases and lignin peroxidases

Table 1: Physico-chemical parameters of chloroperoxidase and lignin peroxidase:


Table 2: Composition of amino acid of chloroperoxidase and lignin peroxidase (in %):

Table 3: Secondary structure prediction of chloroeroxidase and lignin peroxidase (in %):


Table 4: Identification of motifs:

Table 5: Active site Prediction


Yeasmeen Ali et.al Int J Biol Med Res. 2014; 5(3): 4246-4257 Fig 2: Pockets (active site) in motif of chloroperoxidase and lognin peroxidase. 4255


DISCUSSION: Instability index and aliphatic index indicate the relative thermostability of the proteins, whereas GRAVY values show hydrophilic nature of most of them. lignin peroxidases have high percentage of alanine and serine. On the other hand, chloroperoxidases are predominated by leucine, glycine, proline and serine. These amino acids have important role in protein structure. Serine is small in size and hence reasonably common to occur within the tight turns on the protein surface or within the interior of a protein. Serine side-chain hydroxyl oxygen can form a hydrogen bond with the protein backbone [26]. Leucine is a hydrophobic amino acid as it has a branched hydrocarbon side chains usually buried in folded proteins. The hydrophobic effect accounts for stabilization of water-soluble proteins [24]. High percentage of glycine may be responsible for the stability of triple helical structure, since incorporation of large amino acids can cause steric hindrance [27]. Increased proportion of proline residues is significant not only to act as structural disruptor of the secondary structural elements but also to point outward and stabilize the helix [28]. From the CRYSTALP2 result, it is clear that chloroperoxidases are more likely to be crystallized than lignin peroxidases. Among all chloroperoxidase, chloroperoxidase 5 represents maximum propensity for crystallization. The knowledge of crystallization tendency of protein has a great importance in structural biology that provides valuable insight into the structure-function relationship of the proteins [16]. Moreover, crystallographic knowledge of proteins has immense roles in pharmaceutical, biotechnological and chemical industries for rational drug designing, protein engineering and other applications. [29]. Secondary structure analysis showed predominance of random coil over other structures. Large number of random coil gives the protein more flexibility and self-assembly [30]. Protein 3D models were created using homology modeling that were validated by Ramachandran plot. Motif scan tool was used as motif finder in these proteins. Conserved regions of these motifs are important zone to mitigate their activities. It is possible to inactivate these proteins by designing ligands against these conserved sites in 3D protein structure. Moreover, any type of site directed mutagenesis can be performed in these regions to inactivate these proteins. To do so, we have to acquire a deep knowledge on it. cysteine, proline, aspartate, asparagines, histidine, alanine and glycine are major amino acid that not only have structural role but also important in catalytic function. Cysteine is important for disulfide linkage as well as for metal binding [32]. Another important residue is proline. It is unable to adopt a normal helical conformation because it introduces kinks into alpha helices. Aspartate and asparagine are frequently involved in protein active sites. Because of their negative charge, they can interact with positively charged non-protein atoms. Since Aspartate has a shorter side-chain, it is slightly more rigid within protein structures. Thus, it has a slightly stronger preference to be involved in protein active sites [26]. Histidine is the

most common amino acid in protein active sites. They efficiently bind metal ions, often acting together with cysteines. Alanine plays important role in substrate recognition or specificity, particularly in interactions with non-reactive atoms such as carbon. The uniqueness of Glycine [H as R group] also means that it can play a distinct functional role, such as using its sidechain-less backbone to bind to phosphates [26]. Predicted active sites were of similar structure in same protein family. Volume, area and charge density of active sites were satisfactory. All of the chloroperoxidase proteins [except chloroperoxidase 1] share a common ancestral point [marked by arrow, Fig 4] after being diverged from root of the tree. Fungal lignin peroxidase 1 and 3 are more closely related than lignin peroxidase 2. Chloroperoxidase 1 seems to be the outgroup of the tree. Probably this protein has acquired a lot of change in its sequence over time without losing its catalytic activity. CONCLUSION: A number of plants for example crop, fiber or other commercially valuable species are infected by Macrophomina phaseolina fungus and causes a great lose throughout the world. For the pathogenesis of this fungus, different genes are involved. Among these genes, chloroperoxidase and lignin peroxidase are two important genes that are required for a crucial step that is lignin degradation. In present study, we have analyzed physicochemical characteristics, secondary and tertiary structure as well as motifs in 7 chloroperoxidase and 3 lignin peroxidase. During this analysis, we found amino acids like cysteine, aspertate, asparagine, proline, histidine and glycine, which play vital role both structurally and functionally. Moreover, we have searched the active sites in these 10 proteins and found highest pocket area for chloroperoxidase 4 and lignin peroxidase 3. Study of evolutionary relationship reveals that all of the proteins shared a common ancestor. All of these findings help us to understand the characters of these proteins. This will facilitate to design a potent target against chloroperoxidase and lignin peroxidase and protect the fungal susceptible crop species. CONFLICT OF INTERESTS The authors declare that there is no conflict of interests regarding the publication of this paper. References [1]

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